new polyurethane nail lacquers for the delivery of antifungal drugs · 2018-07-31 · nail lacquers...
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UNIVERSIDADE DE LISBOA
FACULDADE DE FARMÁCIA
DEPARTAMENTO DE FARMÁCIA GALÉNICA E TECNOLOGIA
FARMACÊUTICA
New polyurethane nail lacquers for the delivery of
antifungal drugs
Bárbara Susana Gregorí Valdés
Orientadores: Prof. Doutora Helena Margarida de Oliveira Marques Ribeiro
Prof. Doutor João Moura Bordado
Tese especialmente elaborada para a obtenção do grau de Doutor em Farmácia,
especialidade de Tecnologia Farmacêutica.
2017
UNIVERSIDADE DE LISBOA
FACULDADE DE FARMÁCIA
DEPARTAMENTO DE FARMÁCIA GALÉNICA E TECNOLOGIA
FARMACÊUTICA
New polyurethane nail lacquers for the delivery of antifungal drugs
Bárbara Susana Gregorí Valdés
Orientadores: Prof. Doutora Helena Margarida de Oliveira Marques Ribeiro
Prof. Doutor João Moura Bordado
Tese especialmente elaborada para a obtenção do grau de Doutor em Farmácia, especialidade
de Tecnologia Farmacêutica.
Júri:
Presidente:
Doutora Matilde da Luz dos Santos Duque da Fonseca e Castro, Professora
Catedrática e Diretora da Faculdade de Farmácia da Universidade de Lisboa.
Vogais:
- Doutor Francisco Javier Otero Espinar, Titular de Universidad Facultad de
Farmacia da Universidade Santiago de Compostela, Espanha;
- Doutora Maria Eugénia Soares Rodrigues Tavares de Pina, Professora
Associada com Agregação Faculdade de Farmácia da Universidade de
Coimbra;
- Doutora Ana Paula Rocha Duarte, Professora Auxiliar Convidada
Universidade Atlântica;
- Doutor Rui Miguel Galhano dos Santos Lopes, Bolseiro de Pós-Doutoramento
Instituto Superior Técnico da Universidade de Lisboa;
- Doutora Helena Margarida de Oliveira Marques Ribeiro, Professora Associada
Faculdade de Farmácia da Universidade de Lisboa, Orientadora;
- Doutora Joana Marques Marto, Professora Auxiliar Convidada Faculdade de
Farmácia da Universidade de Lisboa.
Fundação para a Ciência e a Tecnologia. Grant (SFRH/BD/78962/2011)
2017
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To my daughter
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“Competition breeds excellence
and whoever says otherwise,
is still hurting from a loss in the past.
Victory is around the corner if you keep competing.”
— Patrick Bet-David
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Table of Contents
Acknowledgements .................................................................................................................. xvi
Abstract ................................................................................................................................. xviii
Resumo ...................................................................................................................................... xx
List of figures ........................................................................................................................... xxv
List of Tables .......................................................................................................................... xxix
Abbreviations & Symbols ...................................................................................................... xxxii
Chapter 1: General Introduction ............................................................................................... 1
1.Onychomycosis ........................................................................................................................ 5
1.1 Epidemiology ................................................................................................................... 5
1.2 Risk factors ....................................................................................................................... 6
1.3 Clinical classification ....................................................................................................... 7
2.Transungual delivery ............................................................................................................... 9
2.1 Nail structure and transungual permeation ....................................................................... 9
2.2 Mathematical description of nail permeability ............................................................... 12
3.Onychomycosis topical therapy ............................................................................................ 13
3.1 Drug delivery enhancers ................................................................................................. 14
3.2 Examples of antifungal drugs ......................................................................................... 20
3.3 Examples of topical pharmaceutical forms .................................................................... 25
4. Nail Lacquer in onychomycosis therapy .............................................................................. 32
5. Polyurethane a potential excipient in therapeutic nail lacquer ........................................... 42
5.1 Synthesis of Polyurethane .............................................................................................. 42
5.2 Monomers for the synthesis of polyurethanes ................................................................ 43
5.3 Methods for synthesis of polyurethanes ......................................................................... 47
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5.4 Synthesis of polyurethanes from carbohydrates ............................................................. 48
5.5 Application of polyurethane in Biomedical Industry ..................................................... 49
5.6 Application of polyurethane in drug delivery system. ................................................... 50
6. References ............................................................................................................................ 53
Chapter 2: Synthesis and characterization of polyurethanes employing carbohydrates ......... 69
1.Introduction ........................................................................................................................... 71
2. Materials and methods ......................................................................................................... 72
2.1 Materials ......................................................................................................................... 72
2.2 Methods .......................................................................................................................... 72
2.2.1 Synthesis of the polyurethane quasi-prepolymers ....................................................... 72
2.2.2 Synthesis of polyurethanes .......................................................................................... 73
2.3 Characterization of polyurethane quasi-prepolymers .................................................... 74
2.3.1 Determination of viscosity .......................................................................................... 74
2.3.2 Free isocyanate content ............................................................................................... 74
2.3.3 FTIR ............................................................................................................................ 74
2.4 Polyurethane characterization ........................................................................................ 74
2.4.1 Solubility studies for polyurethanes ............................................................................ 74
2.4.2 FTIR ............................................................................................................................ 74
2.4.3 NMR ........................................................................................................................... 74
2.4.4 Thermoanalytical measurements ................................................................................. 75
2.4.5 Cytotoxicity assay ....................................................................................................... 75
2.4.5.1 Preparation of sample ............................................................................................... 75
2.4.5.2 Direct contact assay .................................................................................................. 75
3. Results and discussion .......................................................................................................... 76
3.1 Syntheses of polyurethane quasi-prepolymers ............................................................... 76
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3.2 Viscosity and NCO content of polyurethane quasi-prepolymers ................................... 76
3.3 FTIR analysis of polyurethane quasi pre-polymers ....................................................... 77
3.4 Synthesis and characterization of polyurethanes ........................................................... 79
3.4.1 Solubility of PU ........................................................................................................... 79
3.4.2 FTIR analysis of polyurethanes ................................................................................... 80
3.4.3 NMR analysis of polyurethane .................................................................................... 81
3.4.3.1 1HNMR of polyurethane .......................................................................................... 81
3.4.3.2 13C NMR of polyurethanes ....................................................................................... 86
3.5 DSC analysis of polyurethanes ...................................................................................... 86
3.6 Results of cytotoxicity assay .......................................................................................... 87
4. Conclusions .......................................................................................................................... 89
5. References ............................................................................................................................ 90
Chapter 3: Synthesis of biocompatible polyurethanes. Characterization by Techniques
Spectroscopy, GPC and MALDI-TOF ..................................................................................... 95
1. Introduction .......................................................................................................................... 97
2.Material and Methods ........................................................................................................ 98
2.1 Materials ......................................................................................................................... 98
2.2 Methods .......................................................................................................................... 98
2.2.1 Synthesis of quasi-prepolymers .................................................................................. 98
2.2.2 Synthesis of Polyurethanes .......................................................................................... 99
2.2.3 Determination of viscosity .......................................................................................... 99
2.2.4 Determination of free isocyanate ................................................................................ 99
2.2.5 FTIR Characterization ................................................................................................. 99
2.2.6 NMR Characterization .............................................................................................. 100
2.2.7 DOSY ........................................................................................................................ 100
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2.2.8 Hydrodynamic radii (rh) ............................................................................................ 102
2.2.9 Gel Permeation Chromatographic ............................................................................. 102
2.2.10 MALDI-TOF ........................................................................................................... 103
2.2.11 In vitro cytotoxicity assay ....................................................................................... 104
2.2.11.1 Preparation of samples ......................................................................................... 104
3. Results and discussion ........................................................................................................ 105
3.1 Viscosity and free isocyanate of quasi-prepolymers .................................................... 105
3.2 FTIR Characterization .................................................................................................. 106
3.2.1 Characterization of quasi-prepolymers polyurethanes .............................................. 106
3.2.2 Characterization of Polyurethanes ............................................................................ 107
3.3 1H NMR Characterization ............................................................................................ 110
3.3.1 Characterization of some monomer by 1H NMR spectroscopy ................................ 110
3.3.1. 1 Characterization of IPDI ....................................................................................... 110
3.3.1. 2 Characterization of PPG by 1H NMR ................................................................... 112
3.3.1. 3 Characterization of Isosorbide by 1H-NMR .......................................................... 113
3.3.2 Analysis of PUs synthesized of by NMR spectroscopy ............................................ 115
3.3.2.1 Analysis of polyurethanes by 1H-NMR spectroscopy ............................................ 117
3.3.2.2 Analysis of polyurethanes by 13C-NMR spectroscopy .......................................... 121
3.5 DOSY of Polyurethanes ............................................................................................... 125
3.6 Hydrodynamic radii ...................................................................................................... 128
3.7 Result of the analyzis of polyurethanes by GPC .......................................................... 128
3.8 MALDI-TOF ................................................................................................................ 129
3.9 Cytotoxicity assay ........................................................................................................ 131
4. Conclusions ........................................................................................................................ 133
5. References .......................................................................................................................... 134
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Chapter 4: New polyurethane nail lacquers for the delivery terbinafine – formulation and
antifungal activity evaluation ................................................................................................. 139
1.Introduction ......................................................................................................................... 143
2.Materials and methods ........................................................................................................ 144
2.1 Materials ....................................................................................................................... 144
2.2 Methods ........................................................................................................................ 144
2.2.1 Preparation of the PU based nail lacquers ................................................................. 144
2.2.2 In vitro cytotoxicity assay ......................................................................................... 145
2.2.3 Determination of wettability by measurement of contact angle ............................... 146
2.2.4 PALS ......................................................................................................................... 146
2.2.5 SEM ........................................................................................................................... 147
2.2.6 Adhesion test ............................................................................................................. 147
2.2.7 Viscosity measurement ............................................................................................. 148
2.2.8 In vitro release of terbinafine hydrochloride from therapeutic nail lacquers ............ 148
2.2.9 In vitro antifungal activity ......................................................................................... 149
3. Results and Discussion ....................................................................................................... 149
3.1 In vitro cytotoxicity ...................................................................................................... 149
3.2 Wettability .................................................................................................................... 151
3.3 PALS determination of free volume in films ............................................................... 151
3.4 SEM .............................................................................................................................. 154
3.5 In vitro adhesion test .................................................................................................... 155
3.7 In vitro release of terbinafine from Formulations A PU 19-10%, B PU 20-10% and C
PU 21-10% ......................................................................................................................... 156
3.8 Antifungal activity ........................................................................................................ 157
4. Conclusions ........................................................................................................................ 159
5.References ........................................................................................................................... 160
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Chapter 5: Formulation optimization of PU based Nail Lacquers containing Terbinafine
hydrochloride and Ciclopirox Olamine ................................................................................. 165
1. Introduction ........................................................................................................................ 167
2. Material and methods ......................................................................................................... 168
2.1 Materials ....................................................................................................................... 168
2.2 Methods ........................................................................................................................ 168
2.2.1 Preparation of Nail lacquers ...................................................................................... 168
2.2.2 Direct contact cytotoxicity assay for Nail lacquer .................................................... 169
2.2.3 Determination of wettability by contact angle measurement .................................... 169
2.2.4 SEM ........................................................................................................................... 169
2.2.5 Adhesion test ............................................................................................................. 170
2.2.6 Drying time for nail lacquer therapeutics .................................................................. 170
2.2.7 Viscosity determination ............................................................................................. 170
2.2.8 In vitro release of terbinafine from nail lacquers containing different concentrations of
PU ....................................................................................................................................... 170
2.2.9. In vitro antifungal activity ........................................................................................ 171
2.2.10. Final formulations - Permeation studies ................................................................. 171
2.2.11 Porosity measurement ............................................................................................. 173
3. Results and discussion ........................................................................................................ 173
3.1 Direct contact assay ...................................................................................................... 173
3.2 Wettability .................................................................................................................... 175
3.3 SEM .............................................................................................................................. 176
3.4 Adhesion test results ..................................................................................................... 177
3.5 Nail lacquer’s drying time ............................................................................................ 179
3.6 Viscosity values ............................................................................................................ 179
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3.7 Preliminary test for select formulation with better condition to release the terbinafine
............................................................................................................................................ 180
3.8 Antifungal activity ........................................................................................................ 181
3.9 Permeation Test ............................................................................................................ 182
3.10 Porosity study ............................................................................................................. 184
4. Conclusions ........................................................................................................................ 187
5. References .......................................................................................................................... 188
Chapter 6: Conclusions and Final Remarks .......................................................................... 193
Annex 1. Spectrum 1H NMR PEG 1 500 .......................................................................... 201
Annex 2. Spectrum 1H NMR pMDI .................................................................................. 202
Annex 3. Spectrum 1H NMR Sucrose ................................................................................ 203
Annex 4. MALDI-TOF mass spectra of PU19 .................................................................. 204
Annex 5. MALDI-TOF mass spectra of PU20 .................................................................. 205
Annex 6. MALDI-TOF mass spectra of PU21 .................................................................. 206
Annex 7. MALDI-TOF mass spectra of PU22 .................................................................. 207
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Acknowledgements
I would like to express my special appreciation to my advisors. To Professors Helena
Margarida Ribeiro and João Moura Bordado, a special thanks for the opportunity to work in
two excellent group (CERENA and NanoBB). The relentless support and availability, were
crucial for the completion of this work and allowed me to grow as a research.
Also, a special acknowledgement is due to Professors Pedro Manuel Teixeira Gomes, Jose
Ascenço and Paulo Gordo as critical thinking and scientific discussions were crucial for the
completion of this work. To Professor Francisco Javier Otero Espinar for all the support and
trust, as well as for welcoming me into his research group of University Santiago de
Compostela and to Elena Coutrin for all the help with some scientific issues and time
dedicated. To Professor Ana Paula Serro, PhD’s Auguste Rodrigues Fernandes and Clara
Gomes, Centro de Química Estrutural (CQE), Instituto Superior Técnico. Without, your
collaboration a significant part of this work would not be possible.
Furthermore, I would like to mention, my gratitude to PhD. Lídia Gonçalves, for support in
compatibility test. To Professor Humberto Ferreira for the time employ in characterization of
polyurethanes. To Professors Andreia Ascenso and Sandra Simões for the help in validation
process and works in the laboratory. To PhD’s Rui Santos, Margarida Mateus, Susete
Fernandes and Luz Fernandez for the help in chemical characterization and unconditional
support. To MSc. Inês Casais for the graphic desing of some imagen of the text. To PhD
Joana Marto for the precious help in antifungal test. To MSc. Ana Salgado for the help in
developing the HPLC methods.
Thanks so much to Maria Helena Gil for the restless support and friendship.
To the company DS Produtos Químicos Lda., Especially to Mr. Óscar Brás, for the
availability of several raw materials.
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To all my colleagues from the former Nanomedicine and Drug Delivery Systems group for all
the help and constant support. In particular, to Inês Ferreira, Maria Paisana, Paulo Roque
Lino, Sara Raposo, Carla Eleutério, Diogo Baltazar, Diana Gaspar and João Quintas.
An acknowledgement is also due to the Faculdade de Farmácia, Universidade de Lisboa, in
particular the “Departamento de Farmácia Galénica e Tecnologia Farmacêutica”, the Research
Institute for Medicines (iMed.ULisboa), the Microbiological Department and the Chemistry
Department of Instituto Superior Técnico of Universidade de Lisboa for the infrastructures
and conditions that allowed me to develop my PhD work.
To Noel, Susan, my mother and my father for the unconditional love, encourage to finish the
research and support in all the difficult situations.
To the Portuguese government (Fundação para a Ciência e a Tecnologia) for the grant
(SFRH/BD/78962/2011).
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Abstract
Nail fungal infections frequently addressed as onychomycosis present a considerable
prevalence on the global population.
Despite not being a life-threatening disease, this infection causes several social problems to
the infected patients. Unfortunately, the topical therapy available, does not include one of the
most effectively drugs and oral therapy comprises several problems.
Nail lacquers are solutions of film forming polymers, which leave a film on the nail plate after
solvent evaporation. This polymer film can be water resistant or water soluble and can act as a
drug reservoir, allowing a drug to permeate through the nail plate. The nail lacquers water
resistant are occlusive and adhesive to the nail. They present advantages regarding nail
hydration, permanence on the nail plate and patient compliance, when compared to creams,
gels and solutions.
The development of polyurethane based nail lacquers for the delivery drugs to treat
onychomycosis is still a challenge. Nowadays there are drugs with pharmacological activity
(antifungal) for treatment of onychomycosis (i.e. terbinafine hydrochloride), but there is no
nail lacquer formulation in the market capable of delivering them into the nail with success.
Polyurethanes are being used for sustained and controlled delivery of various drugs. The
development of a new polyurethane based on carbohydrates for the delivery of antifungal
(terbinafine hydrochloride and ciclopirox olamine) to treat onychomycosis, is the main aim of
this thesis.
The thesis is divided in two main topics:
The first part presents the synthesis of polyurethanes from carbohydrates and derivatives. The
biocompatible polyurethanes were successfully synthesized employing carbohydrates and
aliphatic isocyanates. The polyurethanes were synthesized by pre-polymerization method.
This process allows maintain better control the structure and properties of the final polymer,
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through a simple change of chain extender. It was possible to obtain polyurethanes with
different properties from the same pre-polymer, that are typically liquids, which facilitates
handling on an industrial scale, when compared with monomeric isocyanate systems. The
polyurethanes were characterized by FTIR, NMR, GPC and MALDI-TOF. The
biocompatibility of these materials was also assessed using keratinocytes cell.
In the second part of our work, four polymers were used to prepare nail lacquers containing
terbinafine hydrochloride as a model drug. The results obtained from the
structural/characterization analysis of these PU based nail lacquers, comprising all the data
and information that allowed the selection of the best of them to optimize the final
formulations.
At this stage, only one of the tested PU was selected to the complete physical, chemical and
microbiological studies with two drug models: terbinafine hydrochloride and ciclopirox
olamine. PU based nail lacquers with different amounts of the polyurethane selected previous
were prepared. Cytotoxicity, wettability, SEM, adhesion test, viscosity, in vitro release and
permeation studies as well as the antifungal activity were performed.
Keywords: Onychomycosis, nail lacquer; polyurethanes; adhesion to keratin, transungual
drug delivery.
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Resumo
As três últimas décadas foram marcadas por significativas alterações nos padrões das
infecções fúngicas. Este fato, aliado ao aumento da população de risco, como doentes com
Síndrome de Imunodeficiência Adquirida (SIDA), transplantados, com patologias
hematológicas ou com outros comprometimentos imunitários, como terapêuticas
imunossupressoras, fez com que estas infecções assumissem uma maior importância no
contexto de saúde pública.
A onicomicose é uma infecção fúngica da lâmina ungueal (unha) que representa entre 15 a
40% de todas as onicopatias. Pode ser provocada por dermatófitos, fungos filamentosos, não
dermatófitos ou leveduras, principalmente as do género Candida. Apesar de estar associada a
diferentes agentes etiológicos, em 90% dos casos os responsáveis são dermatófitos,
principalmente Trichophyton rubrum.
A prevalência das onicomicoses na população em geral tem sido estimada entre 14% e 23%,
sendo que estes números têm aumentado consideravelmente nas últimas décadas e estão
relacionados com a diabetes, doença arterial periférica, psoríase, imunodepressão e
envelhecimento. Em estados avançados, esta patologia pode causar dor, desconforto,
limitações físicas e ocupacionais, condições estas que interferem com a qualidade de vida do
doente.
Os antifúngicos são largamente prescritos na prática clínica para o tratamento de infeções
fúngicas na unha. Os antifúngicos podem ser administrados por via sistémica ou tópica. A
escolha da terapêutica é feita tendo em conta factores como o grau de infeção da unha e
potenciais interações e contraindicações. No entanto, a administração tópica apresenta maior
conforto para o doente.
Para tratamento das onicomicoses (tratamento de iniciais e bem delimitadas), têm sido
usados com muita aceitação desde 1990, sistemas de libertação transungueal baseados em
vernizes medicamentosos. Os vernizes medicamentosos são soluções ungueais, onde se
incluem polímeros, solventes voláteis, promotores da permeação e fármacos. Depois de
aplicado na unha, os solventes voláteis evaporam e os polímeros deixam um sistema matricial
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(filme) onde o fármaco pode estar incluído. Este sistema é formado por cadeias de uma ou de
várias substâncias químicas polimerizadas, que funcionam como agentes moduladores da
permeação. A partir desta matriz o fármaco difunde-se até à estrutura da unha e exerce ação
fungicida. A penetração e o transporte de um fármaco na unha é um requisito para que o
mesmo desempenhe a sua atividade terapêutica. A maioria dos fármacos não consegue
permear a unha, pelo que é necessário um veículo para promover a sua libertação no local de
ação.
Os vernizes medicamentosos preparados a partir de polímeros hidrófobos posuem vantagens
sobre outras formulações. Estes sistemas não são soluvéls em água deixando uma camada de
polímero com maior durabilidade na unha, o que permite manter a sua ação por um maior
período de tempo. O tratamento é simples e indolor, o progresso é visível e permite a remoção
da zona infectada da unha.
Os antifúngicos selecionados nesta tese, como moléculas modelos, foram o cloridrato de
terbinafina (TH) e o ciclopirox olamina (CPX).
O cloridrato de terbinafina, composto sintético derivado da alilamina (C3H5NH2), tem como
mecanismo de ação a inibição da esqualeno epoxidase, uma enzima crucial no processo de
síntese do ergosterol, o que origina a acumulação de esqualeno, levando à disrupção da
membrana com consequente morte da célula fúngica. O cloridrato de terbinafina apresenta
concentrações mínimas inibitórias para o T. rubrum de 0.004–0.06 μg/mL, C. Albicans 0.06-
16 μg/mL e A. fumigatus 1.4 μg/mL. O seu peso molecular é 291.43 g/mol. Trata-se de uma
molécula lipofílica, que possui elevada eficácia no tratamento das onicomicoses por se
difundir muito bem no leito ungueal. Esta molécula ainda não se encontra no mercado em
vernizes medicamentosos.
O ciclopirox olamina é um agente antimicótico sintético da família das piridonas. Possui
amplo espetro com atividade inibitória contra dermatófitos, C.Candida, M. furfur, entre outros
fungos. O mecanismo de ação estudado para a Candida albicans indica que este inibe a
captação de precursores da síntese macromolecular que ocorre em duas etapas: i) inibindo a
entrada de iões metálicos, dos iões fosfatos e do potássio; ii) atuando sobre a cadeia
xxii
respiratória da célula fúngica devido às propriedades quelantes. O ciclopirox olamina
apresenta uma concentração mínima inibitoria para o T. rubrum de 0.031–0.5 μg/mL, C.
Albicans 0.06-0.5 μg/mL e para o A. fumigatus 0.42 μg/mL e um peso molecular 207.03
g/mol. A sua eficácia está relacionada com o fato desta molécula se difundir na epiderme e
nos folículos pilossebáceos, com destaque para a impregnação das camadas superficiais do
estrato córneo. Possui a capacidade de penetrar e atravessar a queratina das unhas. Encontra-
se disponível em verniz medicamentoso e foi utilizado nesta tese como fármaco modelo e
para avaliar a capacidade da utilização de poliuretanos como polímeros de vernizes
medicamentosos.
As formulaçoes de vernizes medicamentosos desenvolvidas nesta fase incluríam solventes
voláteis: etanol, acetato de etilo e butilo. Esta seleção foi realizada de acordo com a pesquisa
efectuada essencialmente em patentes.
Os poliuretanos (PU) são polímeros de grande aplicação industrial, pois apresentam
propriedades físicas e químicas diferentes de acordo com a estrutura dos monómeros que dá
origem ao polímero final. Otto Bayer e colaboradores pela Ferbenindustri na Alemanha, em
1937 foram os primeiros a sintetiza-los. É um material notável por sua alta performance
devido à excelente resistência química, a solventes, à abrasão e à hidrólise, além de possuir
resistência antifungica, e por apresentar dureza e tenacidade combinada com flexibilidade à
baixa temperatura. A ligação uretana (–NH–COO–) é um resultado da reação entre um grupo
isocianato (–NCO) do diisocianato e um grupo hidroxilo (–OH) do poliol.
Os poliuretanos têm sidos aplicados nas indústrias médica e farmacêutica desde 1970 em
próteses e microesferas para regeneração óssea. No entanto, o uso de PU como um excipiente
para a veiculação de farmacos na unha ainda não foi objeto de estudo.
A presente tese está dividida em dois grandes tópicos:
1 – Síntese e caracterização de poliuretanos a partir de carbohidratos.
Esta síntese tem como objetivo a obtenção de polímeros biocompatíveis. Deste modo, os
polímeros foram sintetizados a partir de polipropileno glicol, polietileno glicol, metil difenil
xxiii
diisocianato polimérico, diisocianato de isoforona e de uma fonte renovável, representada
pelos carbohidratos e seus derivados (sacarose e D-isossorbida). Os poliuretanos sintetizados
a partir de sacarose, polietileno glicol e metil difenil diisocianto polimérico deram origem a
polímeros insolúveis em solventes polares, com uma estrutura extremadamente entrecruzada e
não apresentarom biocompatibilidade. A dissolução do polietileno glicol com
dimetilsulfoxido a baixa temperatura de reação contribuí para elevados tempos de síntese e
com a presença do solvente no processo de produção. A proposta para eliminar estes
problemas consistiu em substituir o diisocianto aromático por diisocianatos alifáticos, em
substituir os poliglicolos (sacarose) con glicolos de 2 OH (D-isossorbida) e em substituir o
polietileno glicol por polipropileno glicol.
A síntese de poliuretano a partir de moléculas hidrofílicas com dos hidroxilos livres, permitiu
obter poliuretanos lineares e com solubilidade em solventes polares. A solubilização do
polímero é muito importante porque deste modo é garantido a solubilidade do fármaco na
matriz polimérica. Por outro lado, a síntese de polímeros pouco reticulados permitire obter
uma matriz com um volume livre que permitirá depois a libertação do fármaco. Foram
efetuadas sínteses de poliuretanos a partir de dois isocianatos alifáticos o diisocianato de
isoforona (IPDI) e 4,4' -diciclo-hexil-metileno (HMDI). O resultado destas sínteses,
permitiram obter os poliuretanos obtidos a partir de IPDI mais biocompatíveis quando
comparados com os poliuretanos obtidos a partir de HMDI. Todos os poliuretanos foram
caraterizados por FTIR e RMN para confirmar a sua estrutura. Foram ainda efetuados estudos
dos coeficientes de difusão e volume hidrodinámico, confirmando-se que à medida que
aumenta o peso molecular e a viscosidade, diminui o raio hidrodinámico e o polímero difunde
menos no solvente onde foi solubilizado previamente. Foram determinados os pesos
moleculares por duas técnicas diferentes, Cromatografia de Permeação em Gel e Ionização
por Dessorção a Laser Assistida por Matriz Tempo de Vôo. Ambas técnicas permitiram
concluir que existe uma relação entre peso molecular e relação estequiométrica entre
monómeros que formam parte da estrutura do polímero.
Uma vez sintetizados os poliuretanos com as características ideais, estes foram selecionados
como excipientes de vernizes medicamentosos.
xxiv
2- Preparação de vernizes medicamentosos
O desafio desta fase do trabalho consistiu em obter formulações de vernizes medicamentosos
empregando quatro poliuretanos como excipientes biocompatíveis, seguros, eficazes e com
adesividade a unha como forma farmacêutica com actividade antifúngica.
Para avaliar o melhor polímero, foram preparadas e avaliadas várias formulações de vernizes
medicamentosos contendo cloridrato de terbinafina. Estudos de biocompatibilidade celular
(queratinócitos), determinação do ângulo de contacto entre a unha e os vernizes, o volume
livre das formulações, a morfologia dos filmes bem como a adesão dos vernizes à queratina e
sua viscosidade foram efectuados. Para além destes, foram realizados estudos in vitro de
libertação e de eficácia antifúngica. A selecção do poliuretano foi realizada com base nestes
estudos.
Numa ultima fase, e com o objetivo de optimizar a formulação do verniz medicamentoso, foi
estudado a influência da concentração polímero nos vernizes e a inclusão de ciclopirox
olamina como segundo fármaco modelo.
As formulações desenvolvidas foram caracterizadas avaliadas através de estudos de
biocompatibilidade, de molhabilidade, de microscopia, de adesividade, de viscosidade e de
ensaios in vitro de libertação e de permeação das substâncias ativas bem como de avaliação da
eficácia antifúngica.
Palavras-chave: Onicomicoses, verniz medicamentoso; poliuretanos; adesão à queratina,
sistemas para libertação transungual, ação antifúngica.
xxv
List of figures
Chapter 1
Figure 1. 1. Scanning electron micrograph of nail plate a) no affecting by fungal infection and
b) nail affected by a fungal infection. ........................................................................................ 5
Figure 1. 2. Types of onychomycosis according to the mode and site of invasion of the
pathogen. .................................................................................................................................... 7
Figure 1. 3. Representation of nail structure. ........................................................................... 10
Figure 1. 4. Factors that affect nail permeation of topically applied formulations. Adapted
from (35). ................................................................................................................................. 12
Figure 1. 5. Colloidal carriers for drug delivery. Adapted from (113). ................................... 30
Figure 1. 6. Scheme of synthesis of polyurethane adapted from (141). ................................... 42
Figure 1. 7. Architecture of the chain of polyurethane. Adapted from (142) .......................... 43
Figure 1. 8. Biomedical application of polyurethanes. ............................................................ 50
Figure 1. 9. Release of nanoparticles from polyurethane matrix. Adapted from (162) ........... 51
Chapter 2
Figure 2. 1. Scheme of the quasi-prepolymer reaction. ........................................................... 76
Figure 2. 2. FTIR in ATR mode spectra of the Pre-6, Pre-8, Pre10 and Pre-12. ..................... 78
Figure 2. 3. FTIR in ATR mode spectra of the Pre-14, PEG 1500, DMSO and pMDI. .......... 78
Figure 2. 4. Ideal structure for polyurethanes: a) PUS, b) PUF, c) PUG. ................................ 79
Figure 2. 5. Structure of polyurethanes obtained, a) PUS, b)PUF, c) PUG. ............................ 80
Figure 2. 6. FTIR in ATR mode spectra of polyurethanes prepared with Pre-14. ................... 81
Figure 2. 7. Structure of PEG. .................................................................................................. 82
Figure 2. 8. Structure of pMDI. ................................................................................................ 82
Figure 2. 9. Structure of Sucrose. ............................................................................................. 83
xxvi
Figure 2. 10. Spectrum 1H-NMR of PUS 14/4. ........................................................................ 85
Figure 2. 11. Spectrum 13C-NMR of PUS 14/4 ........................................................................ 86
Figure 2. 12. DSC thermograms of polyurethane PUS 14/4, PUS 14/6, PUS 14/8. ............... 87
Figure 2. 13. HaCaT cells after 72h of proliferation under contact with polyurethane. A) glass
slide (control), B) PUS 14/4. .................................................................................................... 88
Chapter 3
Figure 3. 1. Two step procedure for the synthesis of isosorbide from D-glucose adapted from
(13). .......................................................................................................................................... 97
Figure 3. 2. Typical spectrum DOSY (D (m2s-1 vs δ (ppm)). ................................................ 101
Figure 3. 3. Synthetic route of polyurethane PU19, PU20, PU21, and PU22. ....................... 105
Figure 3. 4. Spectra of quasi-prepolymer polyurethanes. ...................................................... 106
Figure 3. 5. FTIR spectra of Isosorbide, PPG, IPDI and PUs. ............................................... 107
Figure 3. 6. FTIR of PU, C=O, C–H and C–O– C stretching bands of PUs. ......................... 108
Figure 3. 7. FTIR spectrum of PU22. ..................................................................................... 109
Figure 3. 8. The structure of IPDI. ......................................................................................... 110
Figure 3. 9. 1H NMR spectrum of the IPDI (500 MHz, in CDCl3 at room temperature). ..... 111
Figure 3. 10. The structure of PPG. ........................................................................................ 112
Figure 3. 11.1H NMR spectrum of the PPG (500 MHz, in CDCl3 at room temperature). ..... 113
Figure 3. 12. The structure of Isosorbide. .............................................................................. 114
Figure 3. 13. 1H NMR spectrum of the Isosorbide (500 MHz, in CDCl3 at room temperature).
................................................................................................................................................ 114
Figure 3. 14. Segment of polyurethane with monomers IPDI and PPG (A). Segment of a chain
of polyurethane with monomers IPDI and D-isosorbide (B). ................................................ 116
xxvii
Figure 3. 15. Segment of a chain of polyurethane with monomers HMDI and PPG (A).
Segment of a chain of polyurethane with monomers HMDI and D-isosorbide (B). ............. 117
Figure 3. 16. 1H NMR spectra of PUs (500 MHz, in DMSO-d6 at room temperature). ........ 118
Figure 3. 17. 1H NMR spectrum of PU22 in DMSO-d6. Bruker 500 MHz spectrometer. ..... 120
Figure 3. 18. 13C NMR spectra of the polyurethanes (500 MHz, in DMSO-d6 at room
temperature). .......................................................................................................................... 122
Figure 3. 19. 13C NMR spectrum of the PU 22 (500 MHz, in DMSO-d6 at room temperature).
................................................................................................................................................ 124
Figure 3. 20. DOSY spectra of polyurethanes (1H 500 MHz, in DMSO-d6 at room
temperature,0.348 mg/mL). .................................................................................................... 126
Figure 3. 21. Diffusion coefficient vs concentration for PU19‒PU22 in DMSO-d6, measured
at T = 30 oC. ........................................................................................................................... 127
Figure 3. 22. MALDI-TOF mass spectra of polyurethane after deisotoping procedure. ....... 130
Figure 3. 23. HaCaT cells after 72h of proliferation a) glass slide (control) , b) PU19. ....... 132
Figure 3. 24. HaCaT cell viability by MTT after proliferation under different polymers
material. Control (glass slide), (mean ± SD, n = 6). .............................................................. 132
Chapter 4
Figure 4. 1. HaCaT cell viability by MTT after proliferation under different nail lacquer
formulations. Control (glass slide) (mean±SD, n = 6). .......................................................... 150
Figure 4. 2. Scan Electron Micrograph with 1000x magnification. A) dorsal surface of nail
plate B) film of Formulation A PU 19-10%, C) film of Formulation B PU20-10%, D) film of
Formulation C PU21-10%. ..................................................................................................... 154
Figure 4. 3. Release profile of terbinafine from PU based nail lacquer for 24 h in aqueous
solution of 0.5% Tagat® CH 60 at 32ºC (mean ±SD, n = 3). ................................................ 156
xxviii
Chapter 5
Figure 5.1. Micrograph with 400x magnification of HaCaT cells after 72h of proliferation
under contact with different therapeutic nail lacquer: A) glass slide, B) Formulation A PU19-
10% TH, C) Formulation D PU19-15% TH, D) Formulation E PU19-20% TH, E)
Formulation F PU19-25% TH and F) PU19. ......................................................................... 174
Figure 5. 2. HaCaT cell viability by MTT after proliferation under different nail lacquers.
Control (glass slide), (mean ± SD, n = 6)............................................................................... 174
Figure 5.3.Water contact angle of PU terbinafine nail lacquers (mean ± SD, n= 3). ............ 176
Figure 5.4. Scan Electron Micrograph with 1000x magnification. A) dorsal surface of nail
plate B) film of Formulation A PU 19-10% TH, C) film of Formulation D PU19-15% TH, D)
film of Formulation E PU19-20% TH, E) film of Formulation F PU19-25% TH, F) film of
Formulation G PU19-10% CPX and G) Ony-Tec®. .............................................................. 177
Figure 5.5. Nail lacquer adhesion test to keratin of cow horn. .............................................. 178
Figure 5.6. Release of TH from different formulations at 32 ºC (mean ± SD, n = 6). ........... 180
Figure 5.7. Amount of drug determined in nail plate after 11 days of experiments. One-way
ANOVA and Tukey’s multiple comparison tests indicates significant differences between
Ony-Tec® and PU based nail lacquer formulations. ............................................................. 184
Figure 5.8. Cumulative curves of porosity obtained by MIP for treated and untreated nail and
PoreXpert models of the microstructrure of a) untreated nail, b) Ony-Tec® treated nails and c)
Formulation A PU19-10% TH treated nails. Cubes represent the pores and cilinders the
conection between pores. ....................................................................................................... 185
xxix
List of Tables
Chapter 1
Table 1. 1. Overview of the most referenced transungual permeation enhancers. .................. 18
Table 1. 2. Antifungal drug properties that influences permeation. Measurement conditions,
e.g. base/salt used, medium/solvent composition, pH, ionic strength, and temperature are
provided in parentheses when available. Adapted from (37). .................................................. 21
Table 1. 3. Minimum inhibitory concentration for antifungals used in onychomycosis.
(Values displayed in μg/mL, *MIC range). ............................................................................. 23
Table 1. 4. Topical formulation of antifungal. ......................................................................... 25
Table 1. 5. Vehicles composition of CPX nail formulations. Adapted from (115). ................ 31
Table 1. 6. Nail lacquer formulations. ...................................................................................... 35
Table 1. 7. Polyglycols employing in synthesis of polyurethanes. .......................................... 44
Table 1. 8. Name, toxicity and chemical structure of diisocyanates. ....................................... 44
Table 1. 9. Carbohydrates employed as chain extender in the synthesis of polyurethanes. .... 45
Table 1. 10. The most common catalyst in synthesis of polyurethane. .................................... 46
Chapter 2
Table 2. 1. Relation of monomers for synthesis of quasi-prepolymers. ................................... 73
Table 2. 2. Relation of monomers for synthesis of polyurethane. ........................................... 73
Table 2. 3. Formulation, isocyanate content and viscosity of the polyurethane quasi pre-
polymers. (mean±SD, n=3). ..................................................................................................... 77
Table 2. 4. Chemical shifts of PEG 1500. ................................................................................ 82
Table 2. 5. Chemical shifts of pMDI. ....................................................................................... 83
Table 2. 6. Chemical shifts of Sucrose. .................................................................................... 84
xxx
Chapter 3
Table 3. 1. Molar ratios employed in the synthesis of the quasi-prepolymers. ........................ 99
Table 3. 2. Molar ratios employed in the synthesis of the PU. ................................................ 99
Table 3. 3. Values of viscosity and free isocyanate of quasi-prepolymers, (n=3). ................ 106
Table 3. 4. Assignments of 1H NMR and chemical shifts of IPDI ........................................ 111
Table 3. 5. Assignments of 1H-NMR and chemical shifts of PPG. ........................................ 113
Table 3. 6. Assignments of 1H NMR and chemical shifts of Isosorbide. ............................... 115
Table 3. 7. Assignments of 1H-NMR and chemical shifts of PU 19, PU 20 and PU21. ........ 119
Table 3. 8. Assignments of 1H-NMR and chemical shifts of PU 22. ..................................... 121
Table 3. 9. Assignments of 13C NMR chemical shifts for PU19, PU20 and PU21. ............... 123
Table 3. 10. Assignments of 13C NMR and chemical shifts for PU22. .................................. 125
Table 3. 11. Values of D for PU19- PU22, measured at T=30oC, DMSO-d6. ....................... 127
Table 3. 12. Values of rh for PU19-PU22, measured at T=30 oC, DMSO-d6 ......................... 128
Table 3. 13. Average Molecular weight by GPC. .................................................................. 128
Table 3. 14. Higher mass identified by MALDI -TOF and mass calculated according to molar
ratios. ...................................................................................................................................... 131
Chapter 4
Table 4. 1. PU, terbinafine based nail lacquers composition. ................................................ 145
Table 4. 2. PALS parameters (lifetime and intensity components), hole radius (R) and free
volume cavity associated with the o-Ps lifetime for PU, terbinafine based nail lacquers. ..... 153
Table 4. 3 Antifungal activity of different formulations with terbinafine against Trycophyton
rubrum, Candida albicans and Aspergillus brasiliensis. ....................................................... 158
xxxi
Chapter 5
Table 5. 1. Nail lacquers composition. ................................................................................... 169
Table 5. 2. Nail lacquer’s drying time (mean±SD, n= 3). ...................................................... 179
Table 5. 3. Viscosity values of therapeutic nail lacquers (mean±SD, n= 3). ......................... 180
Table 5. 4. Inhibition zone (mm) of all formulations in plate dish for Candida albicans, and
Aspergillus brasiliensis (mean±SD, n=2). ............................................................................. 181
Table 5. 5. Permeation profiles of formulations and Ony-Tec® trough the nail. (mean±SD,
n=3). ....................................................................................................................................... 183
Table 5. 6. Main parameters of the models obtained from porosity data using PoreXpert. ... 186
xxxii
Abbreviations & Symbols
13C-NMR- Carbon Nuclear Magnetic Resonance
1H-NMR- Proton Nuclear Magnetic Resonance
ATCC- American type culture collection
BS- Base
DOSY- Diffusion-ordered NMR spectroscopy
DO- Optical density
CPX- Ciclopirox
CS- HCl salt
DABCO 1,4- Diazobicyclo[2.2.2]- octane
DMDEE -Dimorpholino- diethylether
DMSO- Dimethylsulfoxide
DSC- Differential scanning calorimetry
e.g- Exemple
EW- Hydroethanolic solution.
FTIR -Fourier Transform Infrared Spectroscopy
FWHM- Full width at half maximum
GPC- Gel Permeation Chromatographic
h- hours
HDI- Hexamethylene diisocyanate
HMDI- 4,4′ methylene bis (cyclohexyl isocyanate)
HPLC-High Performance Liquid Chromatography
IPDI- Isophorone diisocyanate
ISO- International Standard Organisation
KC- KCl solution
LD50- Dosis Letal 50%
LDI- L-lysine ethyl ester diisocyanate
MALDI-TOF- Matrix Assisted Laser Desorption Ionization Time Of Flight
MDI- Methylene diphenyl diisocyanate
MIC- Minimum inhibitory concentration
MIP- Mercury Intrusion Porosimetry
xxxiii
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Mw- Molecular weight
NC- NaCl solution
NMR -Nuclear Magnetic Resonance
NS- Nitrate salt
OS-Olamine salt
PALS-Positron Annihilation Lifetime Spectroscopy
PBS- phosphate buffer solution
PEG- Poly ethylene glycol
PPG- Poly propylene glycol
ppm- parts per million
PU- Polyurethane
PUF- Polyurethane from fructose
PUG- Polyurethane from glucose
PUS- Polyurethane from sucrose
rh - Hydrodynamic radius
RSD- Relative Standard Deviation
RT - Room temperature
SEM -Scanning electron microscopy
SEPA®- 2-n-nonyl-1,3-dioxolane
SIF/P- Simulated intestinal fluid
TB- Tris buffer
TH- Terbinafine hydroclhoride
THF-Tetrahydrofuran
UB- Unspecified buffer
UHP- Urea hydrogen peroxide
USP- United Stated Pharmacopeia
W- Water
xxxiv
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
1
Chapter 1: General Introduction
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
2
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
3
This heading section was adapted from a chapter of the book:
Barbara S. Gregorí Valdés, Carolina de Carvalho Moore Vilela, Andreia Ascenso, João
Moura Bordado, Helena M. Ribeiro. Topical Formulations for Onychomycosis. A Review, in
Andreia Ascenso, Helena Ribeiro, Sandra Simões (eds). Carrier-Mediated Dermal Delivery:
Applications. in: the Prevention and Treatment of Skin Disorders. Pan Stanford Publishing.
2016. 503-553.
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
4
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
5
1.Onychomycosis
Onychomycosis is a fungal infection of the nail that causes a great concern because of the
disfiguring appearance of the nails (1). The nail problem is caused by different types of fungi
that can easily attack the nails due to their keratinolytic enzyme (2). In the Figure 1.1 is
showed the aspect of the microorganism and the debased structure of keratin of the nail.
Figure 1. 1. Scanning electron micrograph of nail plate a) no affecting by fungal infection and
b) nail affected by a fungal infection.
1.1 Epidemiology
Onychomycosis prevalence has been reported to occur from 3 to 26% in the general
population (1,3–6).
Onychomycosis cases have been increasing in the past decades, from a prevalence of 1-2% to
14-15% in a 20 year time window (4). According to several large studies, it is expected that
onychomycosis prevalence will increase to 20% or more in the next decade (4).
Onychomycosis is a serious public health problem leading to detrimental physical and
psychological effects. There are specific population groups affected by this disease. About
50% of all psoriasis patients (1-8% of general population) report nail psoriasis and 40 to 60%
are affected by onychomycosis (5,7,8). Onychomycosis also affects about 1/3 of diabetes
patients since they are more prone to suffer foot complications that can lead to dermatophyte
infections, ulceration, osteomyelitis, cellulitis and tissue necrosis that may result at worst in
amputation (9).
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
6
Onychomycosis greatly affects the patients´ quality of life even not being a life threatening
condition. Approximately half of the patients with onychomycosis report some discomfort
due to this condition: pain, difficulty on wearing footwear and walking, emotional
embarrassment and work related difficulties.
1.2 Risk factors
Onychomycosis is an ailment that has several different risk factors. With regard to age, it is
reported that onychomycosis is more prevalent at an older age, being the elderly the most
affected population with an age/infection correlation of 20% and up to 50% of subjects over
60 and 70 years old, respectively (1,10,11). This correlation is often attributed to poor
peripheral circulation, sedentary lifestyle, weaker immune system, diabetes, nail trauma,
difficulty in performing foot hygiene and slower growing nails (2,10,12,13). Children are
rarely affected by onychomycosis, with an incidence of just about 0.4%, since this group has
smaller nail surface, faster nail growth, fewer nail injuries and lower tinea pedis incidence rate
besides less contact with infected surfaces (5,14–16).
Gender is also considered a risk factor being males more often affected by this type of nail
infection (2,11,17). Several authors postulated that this may be due to hormone differences
between sexes, leading to a difference in the capacity to inhibit the growth of dermatophytes
(18). It is also considered that the use of occlusive footwear and nail injuries may contribute
to a higher incidence in males (2).
Recent studies had stated that onychomycosis may have a genetic basis, with an autosomal
dominant pattern of inheritance related to T. rubrum infection, and increased susceptibility
when at least one parent had onychomycosis (5,8,17,19–22).
The environment plays a major role in contracting a fungal infection. The societal factor
cannot be disregarded. Those who regularly does not wear shoes tend to show a lower
infection rate (2,15). On the contrary, athletes display an increased infection rate due to
occlusive footwear which allows a dark and moist environment for onychomycosis to
develop. Also, athletes contact more with moist infected surfaces, like swimming pools,
communal shower rooms and public toilets (23). Nail trauma, synthetic clothing (which
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
7
retains sweat) and tinea pedis are also associated with sports and the incidence of this disease
(2,5,13,21,23–26).
Immunodeficiency in HIV-infected individuals and transplant recipients also consists a risk
factor for onychomycosis. Such persons whose T-lymphocyte count is as low as 400/mm3
tend to have a more widespread infection affecting both toe and fingernails (1,5,11,23,25–29).
Diabetic individuals are three times more likely to have onychomycosis than the general
population, being 34% with onychomycosis. Diabetic people have decreased peripheral
circulation, neuropathy, impaired wound healing and increased difficulty on foot checkups
due to obesity, retinopathy or cataracts. Therefore, injuries in toenails may be unnoticed due
to neuropathy and act as entryways for bacteria, fungi, and other pathogens, leading to serious
complications (4,5,11,21,24,30).
1.3 Clinical classification
There are five types of onychomycosis established according to the mode and site of invasion
by the pathogen: distal and lateral subungual onychomycosis, white superficial
onychomycosis, proximal subungual onychomycosis, total dystrophic onychomycosis (Figure
1.2) and endonyx onychomycosis (1,2,5,29,31–33).
Figure 1. 2. Types of onychomycosis according to the mode and site of invasion of the
pathogen.
Distal and lateral subungual onychomycosis (DLSO)
DLSO is the most common type of onychomycosis (1,2). The fungal infection progresses in
the nail from the distal to the proximal edge, via the distal-lateral margins or through the
lateral nail plate groove, originating from the hyponychium. This type of infection is mainly
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
8
caused by Trichophyton spp. and sometimes by Scytalidium spp. and Candida spp (5). It is
frequent to find paronychia in these individuals, leading to subungual hyperkeratosis, nail
thickening and onycholysis (nail detachment from the nail bed). The subungual space can be
colonized by infectious bacteria and fungi, causing discoloration of the nail plate (1,2,5).
Proximal subungual onychomycosis (PSO)
In this type of onychomycosis, fungi invade the area under the nail cuticle and induce the
infection of the proximal nail plate. Then, it progresses distally along the nail plate. This
clinical manifestation of onychomycosis is mainly prevalent in immunocompromised
individuals, being especially common in AIDS patients where it is considered an early HIV
infection clinical marker. The fungi responsible for PSO are T. rubrum, C. albicans, Fusarium
spp., Aspergillus spp. and Scopulariopsis brevicaulis (5, 25).
Superficial white onychomycosis (SWO)
SWO presents itself as opaque white patches on the dorsal surface of the nail plate. The upper
layers of nail keratin are infected mainly by Trichophyton mentagrophytes and T. rubrum.
Other pathogens responsible for this infection are non-dermatophyte molds such as Fusarium
spp., Acremonium spp. and Aspergillus spp (28).
Endonyx onychomycosis (EM)
EM is the most recently described form of onychomycosis, being reported as an infection of
both superficial and deeper layers of the nail plate. EM does not usually cause nail thickening,
detachment, and inflammatory processes, but it leads to lamellar splitting, coarse pitting and
milky white patches within affected nail plates (4). Usual pathogens are T. soudanense and T.
violaceum (29, 30).
Total dystrophic onychomycosis (TDO)
TDO is usually considered the end stage of any other type of onychomycosis. It can also exist
on its own - primary total dystrophic onychomycosis - occurring mostly in patients with
chronic mucocutaneous candidiasis. It is described as the nearly complete destruction of the
nail plate.
Diagnosing onychomycosis is important in order to guarantee the best possible treatment and
with less side effects. Nowadays, the diagnosis is based on a clinical evaluation in which nail
samples are collected and analyzed by microscopy and fungal culture (1,15,23,25).
Onychomycosis should be differentiated from other similar conditions, such as psoriasis of
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
9
the nail, eczema, bacterial infections, contact dermatitis, traumatic onycho-dystrophies,
chronic onycholysis, lichen planus, chronic paronychia, hemorrhage or trauma,
onychogryphosis, median canalicular dystrophy, pincer nail, yellow nail syndrome, subungual
malignant melanoma and subungual squamous cell carcinoma (4,15). Among these disorders,
it is especially difficult to differentiate onychomycosis from nail psoriasis, since they tend to
share similar nail morphological changes (34).
2.Transungual delivery
All drugs must reach their target location to be more effective. Particularly, the nail unit has
different characteristics from the rest of body surface, and consequently, topical formulations
may not deliver enough drug amounts to the nail bed. Therefore, it is quite important to
clearly understand the nail biology for the development of a more active topical formulations.
2.1 Nail structure and transungual permeation
The nail plate is the main obstacle in the nail unit for drug permeation. It is hard, thin (0.25-1
mm), slightly elastic, translucent, convex shaped, and consists of 80-90 layers of dead,
keratinized, flattened cells tightly bound to each other via intercellular links (35,36). The nail
is constituted by nail plate, lateral fold, proximal fold, matrix, nail bed and distal groove as
showed in Figure 1.3 (37). The nail plate can be divided into three macroscopic layers -
dorsal, intermediate and ventral layers - being the dorsal layer the hardest one (37). Regarding
its chemical composition, the nail is composed of fibrous proteins, keratins (80% “hard”
keratin, remaining “soft” keratin), water and a low amount of lipids (0.1-1%) in contrast to the
stratum corneum (~10%). Under normal conditions, the nail plate can contain 7-12% of water,
but this content can rise to 35% (35–37).
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
10
Figure 1. 3. Representation of nail structure.
Transungual permeation is dependent on drug properties (molecular weight, log P, charge,
pKa, etc.), formulation characteristics (pH, water content, etc.), nail plate properties (diseased
state, hydration, thickness and keratin content) and drug-keratin interactions.
Accordingly, the most significant properties of the permeant drug are:
Molecular weight – it shows an inverse relationship with the drug permeation into the nail
plate. Heavier molecules have a higher time permeation through the keratin network on the
nail plate (35). Drugs below 300 gmol-1 Mw have enhanced transungual permeation rate (37).
Indeed, the molecular weight is considered to be the most significant property affecting the
nail permeation.
Log P – permeation rate was found to decrease with an increase in carbon chain length or
lipophilicity attributed to the hydrophilic nature of the nail plate (37-40).
Charge – non-ionic drugs were found to have about 10-fold greater permeability as compared
to their ionic counterparts (38). However, Elsayed et al. (37) stated that these results did not
take into account the molecular weight of the permeant drug, and regarding other studies,
drug ionization can have a permeation enhancement effect due to an increase of aqueous
solubility maximizing the transungual flux.
pKa and other physicochemical properties – in general, soluble molecules have good
permeation across the nail plate. Weak acidic drugs are well permeated at higher pH, while
weak basic drugs exhibit better permeation at lower pH values. Ionization can also contribute
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
11
to increasing drug solubility. Conversely, if the molecule is sublimable at body temperature, it
will be advantageous for permeating through the diseased nail plate, since it can overcome air
cavities by being sublimed and reach the other side of the cavity (36).
On the other hand, the formulation composition also plays an important role on drug
transungual permeation, as follows:
pH – nail keratin has an isoelectric point of 5.0, turning to a net negative charge at pH 7.4 and
net positive charge at pH 2.0. Therefore, some molecules might be repelled due to charging
depending on pH. Some studies have stated that antifungal drugs have lower activity in acidic
environments (39).
Water content – water enhances the diffusion through the nail plate. As water hydrates the
nail plate, its volume increases, and this swelling causes larger pores, facilitating the
permeation of bigger molecules. (35)
Finally, the nail plate properties influence the drug transungual permeation as well (35,40):
Diseased nail plate – this state has an enormous influence in permeation as diseased nail
plates have uneven thickness, fungi concentration, and onycholysis, which can lead to a
detached nail surrounded by non-detached areas. In some cases, this can be beneficial since
some formulations can be applied to the detached space (41).
Hydration – it increases the ungual permeability of polar compounds, as discussed before.
Drug binding to keratin – it can lead to disappointing results since it reduces the availability
of the permeant drug and weakens its concentration gradient. Although this interaction highly
influences antifungal activity, it is not frequently considered in many studies (42).
In summary, all factors that affect the drug transungual permeation are outlined in Figure 1.4.
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
12
Figure 1. 4. Factors that affect nail permeation of topically applied formulations. Adapted
from (35).
2.2 Mathematical description of nail permeability
Fick’s adapted laws of permeability can be used for studying permeation through the nail
plate.
Flux (J) occurs when an amount of permeant (Q in mol or g) moves across a membrane with a
certain area (A in m2) during time (t in s) (equation A). Fick’s first law describes the flux
where D is the permeant diffusion coefficient (m2s-1) and is the permeant
concentration gradient. The negative sign indicates that the flux is in direction of decreasing
concentration (equation B).
(A) (B)
The permeability coefficient (P) is given by equation C:
(C)
In which K is the distribution equilibrium (distribution constant), and h is the membrane
thickness (in m) (37,43).
According to Kobayashi et al. (44), the functional dependence of D on Mw of the molecule
can be described by equation D:
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
13
(D)
Here D0 represents the diffusivity of a hypothetical molecule having zero molecular weight
and β is a constant.
Nail permeability was found to be independent of the drug lipophilicity according to equation
E (44):
(E) , where β’ = β/2.303
As human nails are not always available for studies, it is important to establish a viable in
vitro model. Animal hooves have been used as a human nail model in the last decades. There
are reports of bovine, porcine and equine hoof use (35). Saner et al. (35) described, the
relationship between permeability coefficients through human nail plates and hoof
membranes can be portrayed by the equation F:
(F)
In which PN is the human nail plate permeability coefficient and PH is the permeability
coefficient through bovine hoof membrane obtained experimentally. Recently researchers
have used keratin films made from human hair as a model for human nails (36,45,46). Other
models used to study nail permeability included wax blocks, nail clippings, cadaver nail
plates and excised cadaver toes (37).
3.Onychomycosis topical therapy
There are several therapeutic options to treat this fungal nail infection: oral, topical and
combined therapy (5,10,15,27,29,47–51). Adhesive patches have also been studied but
displayed very low drug permeation. Thus, these pharmaceutical forms have a low
representatively in the market (52).
Despite being more effective, oral therapy presents several risks for patients, including
adverse reactions and possible drug interactions (10,53–57). This is especially relevant in the
elderly (the most affected age group) who are polymedicated, and therefore, more susceptible
to these reactions (10,12). Thus, it would be expected a higher investment on topical therapy.
Currently, most common topical formulations present a low drug delivery to the infection site
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
14
mainly due to the nail plate characteristics, and the cure rate is lower than oral and combined
therapy. Topical formulations are also bothersome to apply if there are several affected nails,
reducing patient compliance (58,59). Nonetheless, there are several topical formulations
available on the market used in mono or combined therapy. Firstly, it will be reviewed
different drug delivery enhancers, antifungal drugs, and finally, several pharmaceutical forms
employed in the topical treatment of onychomycosis.
3.1 Drug delivery enhancers
The nail plate is a great barrier to drug permeation, as stated before. Thus, it is important to
enhance the permeation to guarantee effective drug delivery.
There are several physical enhancement methods such as nail plate abrasion, etching of the
nail surface with acid, ablation of the nail with pulsed lasers, microporation of the nail plate,
application of low frequency ultrasound and electric current through the nail (38). Although
these methods are effective, reduce patient compliance and need the presence of a specialized
technician to perform them.
Chemical enhancement of ungual drug delivery can also be useful. This type of
enhancement is cheaper, easier to apply, and can be conducted by the patient before or
concomitantly with drug application. It focuses on breaking chemical and physical bonds that
maintain the integrity of the nail plate keratin. Targets for ungual chemical penetration
enhancers are disulfide, peptide, hydrogen and polar bonds. Chemical enhancers are classified
according to the targeted bond and respective mechanism (40).
The nail plate is cohesive due to S-S bonds, and these are great targets for enhancers.
Enhancers targeting this bond will reduce the disulfide linkage, engaging in nucleophilic
attacks as follows:
Nail-S-S-Nail + 2 R-SH 2Nail-SH + R-S-S-R
In this way, enhancers with thiol or ammonia groups with lone pairs of electrons will offer an
advantage in breaking keratin bonding (43).
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Disulfide bond cleaving by reducing agents
Thiols
Thiols are compounds containing sulfhydryl groups (–SH groups) that reduce the disulfide
linkage in the keratin matrix of the nail (60). Examples of thiols used as permeation enhancers
include N-acetylcysteine, mercaptoethanol, N-(2-mercaptopropionyl) glycine, pyrithione and
thioglycolic acid. Once disulfide bonds are cleaved, they are unlikely to be reformed. Thus
these alterations is permanent.
Sulfites
Sodium sulfite is known to cleave disulfide bonds when incubated with proteins and peptides,
producing thiols and thiosulfates. It was then found to enhance transungual permeation both
on pretreatment and co-application (61).
Disulfide bond cleaving by oxidizing agents
In this category of enhancers, hydrogen peroxide has been used alone or in combination.
Pretreatment of nails with hydrogen peroxide has shown to increase mannitol permeation 3-
fold (62). MedNail® technology consists of the pretreating nail with reducing agent TGA,
followed by this oxidizing agent UHP. Treatment with this technology increased terbinafine
drug flux 18-fold (63).
Enhancement by solvents
Water
The effect of water is recognized in the literature as a key enhancer. As nail hydration
increases permeability by a mechanism that is thought to be related to swelling and formation
of larger pores through the keratin matrix, the water content in formulations becomes
necessary to guarantee maximum drug ungual flux. Gunt et al. (64) reported a several fold
increase related to nail hydration. Kobayashi et al.(44) also reported the enhancement of nail
permeation by nail hydration. Notwithstanding, in some cases, nail hydration caused by
formulation does not seem to improve ungual permeation (38).
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DMSO
DMSO is a transdermal enhancer that interacts with the lipid domains of the stratum corneum,
increasing fluidity and promoting partitioning of drugs into the skin. Stüttgen and Bauer (65)
reported that DMSO had enhanced the delivery of econazole. However, DMSO has irritating
properties at high concentrations.
Keratolytic agents
Keratolytic agents disrupt the tertiary structure and hydrogen bonds present in the keratin
matrix, ‘unfolding’ keratin and allowing larger molecules to pass through the nail pores (63).
Urea and salicylic acid soften and hydrate the nail plate, enhancing drug permeation. This
type of agents weakens and damages the nail plate (38). Nevertheless, urea cannot induce
enhanced flux on its own, but it rather acts by synergizing with other enhancers to increase
permeability. For example, urea combined with N-acetylcysteine increased the ungual
concentration of itraconazole by 94-, 20- and 49-fold compared with control (no enhancer),
only urea and only N-acetylcysteine, respectively (38). It was also reported an increase in
permeation for the concomitant use of urea and MPG. This synergy may be explained by
easier access of other permeation enhancers during the keratin unfolding by urea.
Urea has also been used to chemically avulse infected nail at higher concentrations around
40% (66,67). It has also been included in nail lacquers (68).
Enzymes
Keratinolytic enzymes hydrolyze the keratin matrix of the nail plate, altering its barrier
properties and facilitating permeation. Studies have shown that keratinase enzyme markedly
enhances nail permeation (61). This enzyme affects the surface of the human nail, causing
corneocytes to detach and lift off the plate corroding the surface.
Papain is an endopeptidase containing a highly reactive sulfhydryl group and has shown
promising results as a transungual enhancer (35).
Other enhancers
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SEPA®), a skin penetration enhancer, has been reported by Hui et al.(69) to increase the
ungual permeation of econazole. These findings suggest that adding SEPA® to an econazole
lacquer can increase drug permeation up to 6 times, exceeding the MIC necessary to inhibit
fungal growth.
Some formulations contain etching agents which are surface modifiers used to disrupt the
dorsal surface of the nail to enhance permeation and promote adhesion of films. Phosphoric
acid and tartaric acid are two etching agents commonly used as enhancers of transungual
permeation (63). Polyethylene glycols and hydrophobins are other two types of permeation
enhancers (70,71). Curiously, methanol has also shown some advantages in drug permeation
(63).
All the enhancer are summarized in Table 1.1.
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Enhancer Structure Increase in ungual permeation Experiment setup; permeant (Ref)
Thiols/Mercaptans
N-acetylcysteine (5%)
49 x increase in nail drug content Nails immersed in drug solution; Itraconazole
N-acetylcysteine (15%) 2x increase in mean drug uptake, but not statistically significant.
Mean residence time in nail plate increased from 4.2 weeks to 5.5 weeks
Drug was applied twice daily on nail plates of humans
for 6 weeks; Oxiconazole
N-acetylcysteine (3%) 13 x increase in flux from an aqueous formulation; 7x increase from lipidic
formulation Diffusion cells; 5-fluoruracilo
N-acetylcysteine (3%) Drug measurable in presence but not in the absence of enhancer Diffusion cells; tolnaftate
Mercaptoethanol
16 x increase in flux from an aqueous formulation; 8 x increase in flux from lipidic
formulation Diffusion cells; 5- fluoruracilo
Drug measurable in presence but not in the absence of enhancer Diffusion cells; tolnaftate
Thioglycolic acid
3.8x increase in flux Diffusion cells; caffeine
Thioglycolic acid 2 x increase in mannitol concentration in receptor medium Diffusion cells; mannitol
Cysteine
1.7 x increase in mannitol concentration in receptor medium Diffusion cells; mannitol
N-(2-mercaptopropionyl) glycine
2.5 x increase in flux Diffusion cells; water
Table 1. 1. Overview of the most referenced transungual permeation enhancers.
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
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Enhancer Structure Increase in ungual permeation Experiment setup; permeant (Ref)
Keratolytic agents
Hydrogen peroxide 3.2 x increase in mannitol concentration in receptor medium Diffusion cells; mannitol
Urea hydrogen peroxide
18 x increase in flux Diffusion cells; terbinafine
Urea
- Human nail; Fluconazole (68)
Salicylic acid
- (72)
Sulfites
Sodium sulfite
2 x increase in permeation through nail clipping Diffusion cells; 5.6-carboxyfluorescein
Enzymes/Proteins
Keratinase 2.3 x increase in flux Diffusion cells; Metformin (61)
Hydrophobins Up to 3.5x enhancement Diffusion cells; terbinafine (70)
Etching agents
Phosphoric acid
Increase in nail roughness score Human nail (73)
Tartaric acid
Increase in nail roughness score Human nail (73)
Other
Pyrithione
Up to 2.5x increase in flux Diffusion cells; water (60)
2-n-nonyl-1,3-dioxolane (SEPA®)
7x increase in concentration, 1.3x increase in deeper layer flux Diffusion cell; econazole (69)
Table 1.1. Continued.
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter
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3.2 Examples of antifungal drugs
Table 1.2 represents several antifungal drugs and their main physicochemical characteristics
that affect the transungual permeation. It should be noted that permeation coefficient and flux
values were not included as reported in the literature considering that there are different
methods and protocols to determine them (37). Since these coefficients are dependent on
many factors, it is more appropriate to understand their values in the literature context.
The capability of inhibiting fungal proliferation is usually measured by assessing the MIC of
the drug (74,75). MIC values for antifungal drugs are summarized in Table 1.3. Although
these values do not take into account some characteristics (keratin binding, pH influence),
they can serve as guidance (37).
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Table 1. 2. Antifungal drug properties that influences permeation. Measurement conditions, e.g. base/salt used, medium/solvent composition, pH, ionic
strength, and temperature are provided in parentheses when available. Adapted from (37).
Antifungal Drugs Chemical Structure Mw
(g/mol) Aqueous solubility (mg/ml) Log P
log n-octanol/aqueous medium
distribution constant pKa Ref
Amorolfine
317.5
9.320 (CS, W, T20)
9.995 (CS, W, T32)
8.8 × 10− 3 (CS, pH 7.4 PB, T32)
5.7 0.33 (CS, W, T20) 6.6 (37,76)
Bifonazole
310.4 0.13 × 10− 3 (BS, pH 7.4 PBS, T32)
0.51 × 10− 3 (BS, pH 8–10 0.1 M NC, T25)
0.345 (BS, I = 0.02 M pH 7.0 UB, T25)
4.8 4.77
5.2 (BS, 10 mM pH 7.4 PB, RT)
5.85 (I = 0.01 M UB, T25)
5.72 (0.1 M NC, T25)
(37,76)
Ciclopirox
207.3
12.4 (OS, W, RT)
8.590 (OS, W, T32)
1.47 (OS, 5 mM pH 7.0 TB, RT) 1.020 (OS, pH 7.4 PB, T32)
0.22 (OS, 0.1 M HCl, RT)
2 0.53 (OS, 5 mM pH 7.0 TB, RT)
7.2
8.07 ± 0.05 (42% v/v EW, T32)
(37,76)
Clotrimazole
344.8 2.7 × 10− 3 (BS, pH 7.4 PB, T32)
0.39 × 10− 3 (BS, W, T25) 5 4.9 (BS, 10 mM pH 7.4 PBS, RT)
6.02 ± 0.05 (0.15 M
KC, T25)
4.74 ± 0.04 (42% v/v
EW, T32)
(37,76)
Efinaconazole
348.4 Predicted: 0.61 2 - Predicted:7.45-12.7 (37,76)
Fluconazole
306.3 5 (BS, W, T23)
14 (BS, 0.1 M HCl, T23) 0.4 0.5 (BS, 100 mM pH 7.4 PBS)
1.76 ± 0.10 (0.1 M NC, T24)
(37,76)
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Table 1.2. (continued).
Antifungal
Drugs Chemical Structure
Mw
(g/mol) Aqueous solubility (mg/ml) Log P
log n-octanol/aqueous medium
distribution constant pKa Ref
Itraconazole
705.63 0.00964 5.7 - 3.70 (37,76)
Luliconazole
354.3 0.00062 (BS, W) 4 4.34 (pH 7.16 UB, T20)
3.78 (pH 4.00 UB, T20) 4.65 (37,76)
Tavaborole
151.9 ~ 1.0 1.24
-
- (37,76–78)
Terbinafine
291.43
2.92 (CS, pH 3 UB, T25)
1.57 (CS, pH 3 W, T25)
1.12 (CS, pH 5 W, T25)
0.101 (CS, pH 5 UB, T25)
0.02 (CS, pH 6.8 SIF/P, T37)
1.5 × 10− 3 (CS, pH 9 UB, T25)
5.6
6.0 ± 0.1 (neutral form, 0.15 M KC, T37) [50]2.3 ± 0.1 (ionized form, 0.15
M KC, T37)
5.5 ± 0.1 (calculated for pH 6.8 from the
two above-mentioned values, T37)
7.05 (0.15 M KC, T37) (37,70,76)
Tioconazole
387.7 0.0165 5.3 4.4 6.77 (76)
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Table 1. 3. Minimum inhibitory concentration for antifungals used in onychomycosis. (Values displayed in μg/mL, *MIC range).
MIC (μg/mL) Amorolfine Bifonazole Butenafine Ciclopirox Efinaconazole Itraconazole Ketoconazole Tavaborole Terbinafine
Der
ma
top
hy
tes
T. rubrum 0.004-0.015* ≤0.12-1* 0.015-0.12* 0.031-0.5* ≤0.001–0.015* ≤0.015-0.125* 0.06-0.5* 1-2 0.004-0.06*
T. mentagrophytes 0.004-0.06* 0.5-4* 0.06-0.12* 0.031-0.5* ≤0.001–0.03* ≤0.015-0.5* 0.5-2* 2 0.004-0.5*
T. tonsurans 0.25 0.5-2* 0.12 0.25 0.016 0.06-0.25* 0.5-2* 0.015-0.06*
T. verrucosum 0.12 0.25 0.12 0.13 0.0039 0.12 0.12 0.015
M. canis 0.06-0.25* 1-2* 0.12 0.25 0.13-0.25* ≤0.015-0.03* 0.25-0.5* 0.008-0.03*
M. gypseum 0.063-0.25* ≤0.12-8* 0.06-0.12* 0.31 0.010 0.031-0.25* 0.5-4* 0.004-0.06*
E. floccosum 0.13-0.25* 0.25-0.5* 0.06 0.31 0.005 0.03-0.5* 0.25-0.5* 1 0.015-0.06*
T. interdigitale 0.017
No
n-d
erm
ato
ph
yte
s
Geo. candidum 1-2* >64 >0.5 1 2-4* 0.12-0.25*
Scopulariopsis brevicaulis 0.09 2-4 0.5 0.59 0.25 >4 2-4* 0.12
Aspergillus fumigatus >4 2-4* >0.5 0.42 0.089 0.5-1* 8 0.25 1.4
Fusarium oxysporum >4 >64 >0.5 1 1 >4 >8 >2.5
Fusarium solani 4 >64 >0.5 >4 0.5 >4 >8 2 4
References (79,80) (79) (79) (80,81) (80) (79,80) (79) (77,78) (79,80,82)
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
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Table 1.3. (Continued).
MIC (μg/mL) Amorolfine Bifonazole Butenafine Ciclopirox Efinaconazole Itraconazole Ketoconazole Tavaborole Terbinafine
No
n-d
erm
ato
ph
yte
s
Fusarium
verticillioides
>64 >64 >0.5 2 2 >0.5
Acremonium
potronii
0.26 0.25 0.31 2.5 0.25
Acremonium
sclerotigenum
1 1.4 0.18 4 0.09
Aspergillus
flavus
4 3.4 0.11 0.18 0.11
Aspergillus niger 4 0.63 0.2 0.63 0.16
Aspergillus
sydowii
4 0.59 0.037 0.3 0.076
Aspergillus
terreus
4 0.5 0.09 0.21 0.13
Aspergillus
nidulans
4 1 0.0078 0.089 0.063
Yea
sts
C. albicans 0.03-0.5* 0.06-0.5* 0.0005-0.25* 0.004-2* 0.5 0.06-16*
C. glabrata 4.9 0.13 0.026 0.74 8
C. krusei 0.27 0.21 0.024 0.38 8
C.parapsilosis 0.56 0.22 0.0046 0.13 0.28
C. ropicalis 4 0.5 0.014 0.31 8
C.guilliermondii 0.25 0.25 0.016 0.13 1
C. kefyr 0.063 0.13 0.002 0.031 2
C. lusitaniae 0.5 0.25 0.0039 0.13 4
References (79,80) (79) (79) (80,81) (80) (79,80) (79) (77,78) (79,80,82)
New polyurethane nail lacquers for the delivery of antifungal drugs Chapter 1
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3.3 Examples of Topical Pharmaceutical Forms
Antifungal drugs are formulated in several topical pharmaceutical forms, such as creams, solutions, gels and lacquers (Table 1.4).
Table 1. 4. Topical formulation of antifungal.
Antifungal Chemical
Group
Commercial
name
Approved in Fungals Formulation Posology Effectiveness Reference
Amorolfine Morpholine Loceryl,
Locetar, Sinibal,
Unglyfol,
Curanail
UK (1991), Switzerland
(1991), France (1992),
and more than 50 other
countries Not approved
in USA and Canada
Candida sp,
Trichophyton sp,
Microsporum sp,
Epidermophyton
5% lacquer 1-2 weekly, 6-
12 months after
removal of
affected area
50%
mycological
cure in cases
of distal
fingernail and
toenail
onychomycos
is
(37,83–91)
Bifonazole
(+40% urea)
Imidazole Canespor,
Canesten,
Mycospor,
Amypor
(Onyster,
Canespro)
Australia, Portugal, other
countries
T. rubrum, T.
mentagrophytes,
T. tonsurans, T.
verrucosum, M.
canis, M.
gypseum, E.
floccosum ,
Scopulariopsis
brevicaulis,
Aspergillus
fumigatus
1% cream
(40% urea)
After ablation
of the nail with
40% urea
cream,
application
once daily for 4
weeks
33.6% overall
cure rates
(37,66,67,77,8
8–90)
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Table 1.4. (Continued)
Antifungal Chemical
Group
Commercial
name
Approved in Fungals Formulation Posology Effectiveness Reference
Ciclopirox Hydroxypyri
done
derivative
Mycoster,
Niogermos,
Batrafen, Kitonail,
Ony-Tec,
Stieprox, Loprox,
Penlac, Ciclopoli,
RejuveNail
France (1991), Germany
(1992/2008), Austria (1999),
Canada
Trichophyton sp, S.
brevicalis, Candida
sp, Malassezia
furfur, Aspergillus
sp, Fusarium solan
8% lacquer Once daily, 48
weeks
34-46%
mycological
cure
29-36% overall
cure
(37,77,83,88–90,92–
94)
Efinaconazole Triazole Jublia Canada (2013), USA
(2014)
T. rubrum, T.
mentagrophytes,
Aspergillus sp,
Candida albicans
10% topical
solution
Once daily for 48
weeks
53-55%
mycological
cure rate, 15-
18% complete
cure
(37,87–90,95)
Tavaborole Oxaborale
antifungal
Kerydin USA (2014) T. rubrum, T.
mentagrophytes,
Aspergillus sp,
Fusarium sp,
Candida albicans
5% topical
solution
Once daily for 48
weeks
31-36%
mycological
cure, 6.5-9.5%
complete cure
(37,77,87–
90,96,97)
Tioconazole Imidazole Trosyl, Trosyd Portugal, UK, France and
30 more countries
T. rubrum, T.
mentagrophytes
28% solution Daily, 3-12
months
22% of
patients
mycological
cure
(3,37,83,88–90,98)
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Cream
Creams are semi-solid topical preparations used for delivery of one or more active substances,
or for their emollient or protective action. The base may consist of natural or synthetic
substances, being a multiphase preparation with lipophilic and aqueous phases. Lipophilic
creams have the lipophilic phase as the continuous one, containing water-in-oil emulsifying
agents at a higher percentage. The opposite is observed in hydrophilic creams. Creams may
also contain other suitable excipients, such as preservatives, antioxidants, stabilizers,
emulsifiers, thickeners and penetration enhancers (99–101).
As reported by Tietz et al. (67), a 1% bifonazole cream used after nail ablation by 40% urea
paste (Canespor®, Canespro®) was effective in the treatment of onychomycosis. Bifonazole
is an imidazole antifungal drug, acting by blocking the conversion of 24-
methylendihydrolanosterol to desmethylsterol in fungi as well as inhibiting HMG-CoA (3-
hydroxy-3-methylglutaryl-coenzyme A) and compromising the cell membrane. It is active
against most dermatophytes, some non-dermatophytes, and molds. This bifonazole cream also
contained benzylic alcohol, cetostearyl alcohol, cetyl palmitate, octyldodecanol, polysorbate
60, sorbitan monostearate and purified water (102). The urea paste was applied daily with an
impermeable bandage until the ablation of the nail was achieved, and then 1% bifonazole
cream was applied once daily for 4 weeks. At the end, 33.6% of patients were cured
(55,66,67,79,88–90).
Lahfa et al. (66) studied the efficacy of using bifonazole and urea in the same formulation
(Amycor Onychoset®).
Other cream formulations in the European market include Selergo®, Loprox®, Ertaczo®,
Avage®, Mycoster®, Locetar® and Fougera® (35,88,89).
Solution
Topical solutions are liquid preparations used for the delivery of one or more active
substances that are solubilized in a suitable vehicle. These formulations are transparent and
free of visible particles. They may also contain preservatives, antioxidants, stabilizers and
buffers (99–101).
Solutions have some advantages compared to other topical formulations (41,42,103). As a
liquid solution, the formula can be applied to the stratum corneum and subungual space as
well as on the nail plate surface, and it does not require the patient to posteriorly remove the
film as necessary for a nail lacquer (103).
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In the USA market (42,96,104–107), a 10% efinaconazole solution has been recently
available. This formulation contains 100 mg of efinaconazole per gram and its inactive
ingredients are alcohol, anhydrous citric acid, butylated hydroxytoluene, C12-15 alkyl lactate
(co-solvent), cyclomethicone (wetting agent), diisopropyl adipate (co-solvent), disodium
edetate and purified water. This formulation should be applied once daily for 48 weeks.
Overall cure rates were reported in 15-18% of patients, being mycological cure rates between
53-56% of patients studied. Efinaconazole acts by inhibiting the conversion of lanosterol to
ergosterol in the ergosterol synthesis pathway. It possesses a broad spectrum of activity
against dermatophytes, non-dermatophytes and yeasts (16,23,37,77,80,83,87,108,109).
5% Tavaborole solution (Kerydin®) is also available in the United States of American
market, and it contains ethanol USP, propylene glycol USP and edetate calcium disodium
USP (41,77). This solution should be applied once daily for 48 weeks. In conducted clinical
trials, 31-36% mycological cure and 6.5-9.5% complete cure was achieved in patients treated
with this drug. It shows activity against yeasts, molds, dermatophytes and filamentous fungi.
It was specially developed for onychomycosis treatment (96,97).
There is a 28% tioconazole topical solution in the European market (Trosyd®, Trosyl®)
formulated with undecylenic acid and ethyl acetate (110). It is applied daily for 3-12 months.
The average overall cure rate is 22%. Nowadays, its use is in decline as other more effective
antifungal drugs appear on the market (83).
Naumann et al. (111) describe several formulations, including a topical solution for the use of
EV-086K, a new antifungal with a high lipophilicity which is not beneficial to transungual
delivery (111). This solution was formulated by dissolving 5.7% of EV-086K in 30% ethanol
and 63.75% water, besides the presence of Transcutol®P as solubilizer, 0.05% each of citric
acid and sodium phosphatemonobasic for pH adjustment and 0.1% of butylated
hydroxytoluene and, EDTA for chemical stability. The study showed that this formulation
provided high permeability at the beginning of the penetration process (test in equine hoof),
but the maximum drug concentration stabilized at 35% in the acceptor phase after 24h. These
results were not significant compared to other formulations.
Other topical solutions available in the market are Canespor®, Mycoster®, and Lamisil®
among others (88).
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Gel
Gels are formed by suitable gelling agents and can be classified as lipophilic gels or
hydrophilic gels. Lipophilic gels (oleogels) usually consist of liquid paraffin with
polyethylene or fatty oils gelled with colloidal silica, aluminum or zinc soaps. Hydrogels
(hydrophilic gels) are prepared with water, glycerol or propylene glycol gelled with gelling
agents such as starch, cellulose derivatives, carbomers and magnesium-aluminium silicates
(100).
Although there are some gel formulations in the market (e.g. Lamisil®, a terbinafine
formulation), they are mostly used for skin fungal infections instead of onychomycosis.
Along with the cream formulation previously referred, Naumann et al. (111) also studied a gel
formulation for EV-086K delivery. Hydroxyethyl cellulose gel was formulated with the
following excipients: 10% of EV-086K, 1.5% glycerol, 1% Tylose and 87.5% water. Among
other tested formulations, the hydrogel had the less promising results in equine and bovine
hoof penetration test (only 22% and 15% of applied dose was recovered, respectively), and
thus it was not evaluated in the human nail.
Kerai et al. (53) have recently investigated the use of UV-curable gels (currently used in
cosmetics) for the treatment of onychomycosis. The formulated gels contained diurethane
dimethacrylate, ethyl methacrylate, 2-hydroxy-2-methylpropiophenone, an antifungal drug
(amorolfine HCl or terbinafine HCl) and an organic liquid (ethanol or 1-Methyl-2-
pyrrolidone) as drug solvent. Amorolfine was released to a greater extent than terbinafine, and
even not reaching concentrations as high as the comparative nail lacquer, the concentrations
were well above the MIC for T.rubrum, the main pathogen for onychomycosis. In addition,
cured gel formulation could maintain the drug release for longer periods, being a promising
candidate for future onychomycosis treatment.
Advances in Nail Formulations
Colloidal Carriers
The literature reports the success of some colloidal carriers for onychomycosis treatment
since the drug easily diffuses along the skin tissue to the nail bed. Therefore, application to
the skin surrounding the nail could be an effective area to treat for onychomycosis (112–114).
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Colloidal carriers or colloidal drug delivery systems are particulates or vesicular dosage
forms, having a size range from 1 nm to 0.5 μm (Figure 1.5). They are essential for successful
drug transport and delivery by protecting and maintaining the loaded drug until the site of
action is reached.
Figure 1. 5. Colloidal carriers for drug delivery. Adapted from (113).
Along with gel and solution formulations, Naumann also studied a colloidal carrier
formulation for the delivery of EV-086K, containing water, propylene glycol, emulsifiers
(Tagat®O2V and Synperonic™PE/L 101) and an oil component (Pelemol® BIP). This
colloidal carrier system contributed to recovering 7.25 ± 0.30% drug from the nail slices.
These results were comparable to those obtained with a tested solution and nail lacquer (111).
Nogueiras-Nieto et al. (115) developed in 2013 an aqueous formulation based on
polypseudorotaxanes of Pluronic® F-127 and ciclopirox complexed with partially
methylated β-cyclodextrin. The obtainment of in situ gelling thermosensitive hydrogels was
due to the presence of the poloxamer Pluronic ®F127 (PF127), which facilitated drug
solubilization via micelle creation forming a gel upon nail application. Partially mβ-CD was
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also added to further improve drug solubilization. In addition, a penetration enhancer (N-
acetyl-l-cysteine) alone or in combination with urea was also added to the formulations.
Ciclopirox was incorporated into three different vehicles–simple aqueous solutions,
thermosensitive hydrogels, and polypseudorotaxanes thermosensitive hydrogels. The
composition of these vehicles is presented in Table 1.5.
Table 1. 5. Vehicles composition of CPX nail formulations. Adapted from (115).
Recently Yang et al. (113) have developed a gel with transfersomes containing terbinafine
prepared by ethanol injection method. The final formulation contained terbinafine,
phospholipids, polysorbate 80, sodium cholesteryl sulfate, anhydrous ethanol, sodium
benzoate, sodium pyrosulfate and phosphate buffer (pH 5.0). After 12h, it was obtained 88.52
± 4.06 mg.cm-2 and 94.38 ± 5.26 mg.cm-2 of permeated and penetrated drug, respectively.
Vaghasiya et al. (116) studied another formulation composed of solid lipid nanoparticles for
sustained release and skin targeting of terbinafine for the treatment of onychomycosis. The
terbinafine loaded solid lipid nanoparticles formulation consisted of Compritol® 888 ATO as
lipid matrix, Pluronic® F-127 as a stabilizer and distilled water as a dispersion medium, and
prepared by the solvent injection technique. It was observed 40.57 ± 1.76% of the applied
drug retained in the skin after 8h (116).
Terbinafine in transfersome 067 is a carrier -based liquid spray that has been developed for
the delivery of terbinafine to the nail bed to treat onychomycosis. A 2011 study demonstrated
that Terbinafine in transfersome 067 had greater antifungal activity against onychomycosis
caused by dermatophytes compared to the free form vehiculated in an oral formulation (117).
This formulation has also been tested in clinical trials (118).
Name Solvent AC
(%)
PF127
(%)
Mβ-
CD
(%)
CPX–N-acetylcysteine solution (CPX–AC) Water 10 – –
CPX–thermosensitive hydrogel (CPX–TH) Water 10 20 –
CPX–polypseudorotaxanes thermosensitive
hydrogel (CPO–PPR)
Water 10 20 10
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There is another study with terbinafine -loaded liposomes formulation composed of
bioadhesive polymers, pullulan, and Eudragit®L100, and prepared by thin film hydration
method. This formulation also showed interesting results (119).
Barot et al. (120) studied a microemulsion-based gel containing terbinafine, oleic acid,
Labrasol, Transcutol, water and Carbomer 934P. This formulation was developed to be
applied between the nail bed and nail plate generated by onycholysis. It was obtained 49.3% ±
4.12% drug retention in the skin, and the total amount of terbinafine permeated after 12h was
244.65 ±18.43 μg.cm−2. In 2012, Barot also described a microemulsion based antifungal gel
incorporating itraconazole (120).
Barot et al. (121) developed a gel microemulsion containing itraconazole for the topical
treatment of onychomycosis. The microemulsion contained benzyl alcohol, isopropyl
myristate, Pluronic F68 (surfactant), ethanol, double distilled water and itraconazole. The
microemulsion was incorporated into a gel by adding Carbomer 934P. Nail permeation
enhancers like urea and salicylic acid were also used to increase drug penetration through the
nail plate. The optimized formulation showed promising results: 92.75% drug entrapment
efficacy and a complete drug release in 60 min with a highest nail uptake of 0.386%/mm2 (39
mg drug) (72).
Angamuthu (122) described the study of poly (lactide-co-glycolide)) microspheres for the
controlled release of terbinafine administered by intralesion injection. These microspheres
were developed using O/W emulsification and modified solvent extraction/evaporation
technique with methylene chloride, and methanol. This formulation achieved controlled
release through 30 days and proper deposition onto the nail bed and plate.
Another microemulsion-based gel was studied by Kumar et al. (123). This gel contained
fluconazole (against Aspergillus niger), oleic acid, polysorbate 80, propylene glycol and
water. It exhibited an in vitro drug release of 72.23% in 7h.
4. Nail Lacquer in onychomycosis therapy
Nail lacquers have been used for a long time in cosmetics, with both protective and decorative
purposes. In the 1990’s decade, nail lacquers started being used to administer drugs to the nail
unit. Nail lacquers are solutions of film-forming polymers which leave a polymer film on
the nail plate after solvent evaporation. This polymer film can be water resistant or water
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soluble and acts as a drug reservoir, allowing it to permeate through the nail plate. Depending
on the formulation and drug, the lacquer can be removed either mechanically or with organic
solvents after a certain time, and it should be reapplied to reestablish the drug pool. Thus, the
duration of the film residence in the nail constitutes an important property of a lacquer
formulation (124). Although these formulations are commonly applied with a brush, there are
also other types of applicators such as spatulas and sponge tips.
Being occlusive and adhesive to the nail, nail lacquers’ films have advantages regarding nail
hydration, permanence on the nail plate and patient compliance (when compared to creams,
gels, and solutions).
These preparations usually contain the intended drug, a polymer, a volatile solvent and
suspension agents (56,91,125).
The drug should present the following properties: a) low MIC for pathogens causing the
disease (dermatophytes, yeasts, etc.), being effective at low concentrations; b) low molecular
weight and volume to better permeate through the keratin matrix pores; c) water soluble, since
water easily permeates through the matrix vehiculating the drug; and d) affordable and easily
obtainable, so that the nail lacquer can be easily produced and obtained by patients (61).
The polymer choice can also influence the nail lacquer quality. Hydrophilic polymers have
advantages regarding permeation and adhesion to the nail plate, having a soft, flexible and
matte finish, which can improve patient compliance. However, these polymers allow more
drug loss and are less occlusive. Hydrophobic polymers have a more durable, harder and
glossier finish. This contributes for a more occlusive lacquer which can compromise the
cumulative amount of permeated drug. Employing both types of polymers seems to be a good
approach since it will combine both excellent permeation characteristics and higher resistance
to environmental drug loss (126).
Effective formulations usually include permeation enhancers such as thiols/mercaptans,
keratolytic agents, keratinases, etc. Other excipients can also be critical to allow better
permeation conditions, including hydration, pH, and solubility. This can be achieved by using
plasticizers, humectants, solvents, solubilizers, etc (127).
Colorless and non-glossy medicated nail lacquers are more acceptable by male patients (39).
Despite male patient compliance, it could be considered advantageous for female patients to
formulate a colored medicated nail lacquer for the treatment of onychomycosis since this
disease alters the normal appearance of the nail.
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After screening for the most suitable formulation components, it is important to check their
compatibility and progressively adjust the quantities through pre-formulation studies.
There are several parameters to be evaluated in nail lacquer formulations to assess a higher
quality, such as drug content, drug permeation studies (permeation coefficient, flow, the
cumulative amount of permeated drug); gloss; flow; film adhesivity; viscosity; pH; drying
time; non-volatile content, etc. According to this quality control, nail lacquers must present
the final properties: a) be physically and chemically stable; b) release therapeutic levels of
drug onto the nail; c) have a suitable viscosity to freely flow into all the edges and grooves of
the nail for easy application; d) dry quickly (3-5 minutes) and form an even film once applied;
e) adhere to the nail plate to not come off or flake during daily activities, but at the same time,
be easily removed with an enamel remover and f) be cosmetically pleasant and well tolerated
(38).
Several examples of nail lacquer formulations for onychomycosis treatment are presented in
Table 1.6. However, only nail lacquers containing amorolfine and ciclopirox are currently
commercialized (39).
Amorolfine is a morpholine antifungal agent approved for the treatment of onychomycosis in
1981 (128). It is usually presented as a nail lacquer containing 5% amorolfine. This active
substance inhibits delta14 reductase and delta7-delta8 isomerase, causing ergosterol depletion
and ignosterol accumulation in the cytoplasmatic membrane of the fungus cell. Amorolfine is
effective against dermatophytes (Trichophyton spp., Microsporum spp., Epidermophyton
spp.), yeasts (Candida spp., Cryptococcus spp., Malassezia spp.) and some molds (Alternaria
spp., Hendersonula spp., Scopulariopsis spp.). Amorolfine can be present in the nail up to
27% from the nail lacquer formulation after 2 weeks treatment, being this concentration
enough to inhibit most fungi. This formulation should be applied once or twice weekly for 6
months. Studies report mycological cures in 52-60% of the patients, and complete cures (both
clinical and mycological) up to 44% of patients (54,77). Even after apparent cure, some
authors recommend the use of amorolfine as prophylaxis (129). Amorolfine is available in
Europe (e.g. Loceryl®, Locetar®, Curanail®), but not in USA (77,89–91,130).
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Table 1. 6. Nail lacquer formulations.
Drug Commercial name Polymer Solvents & other excipients Ref.
Econazole
Butyl methacrylate,
dimethylaminoethyl
methacrylate, methyl
methacrylate polymer
2-n-nonyl-1,3-dioxolane
Ethanol (69)
Ciclopirox
Hydroxypropyl chitosan (HPCH)
Cetostearyl alcohol
Ethyl alcohol (95°)
Ethyl acetate
Purified water
(131)
Ciclopoli®
Hydroxypropyl chitosan (HPCH)
Ethyl acetate
Ethanol 96%
Cetostearyl alcohol
Water
(126)
Pluronic F127
Partially methylated β-cyclodextrin
N-acetyl-l-cysteine (115)
Mycoster®
Methylvinyl ether
Ethyl acetate
Isopropanol
Maleic acid monobutylester
(128)
Fluconazole
Polyvinylpyrrolidone K25
Glycerol triacetate
Docusate sodium
Urea
Ethanol
Demineralized water
(68)
Tavaborole
Poly (vinyl methyl ether alt maleic acid
monobutylester) Ethanol
(78)
Poly (2-hydroxyethyl methacrylate) Ethanol
Dibutyl sebacate
Water
Poly (vinylacetate) Ethanol
Ethyl acetate
Dibutyl sebacate
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Table 1.6. (Continued)
Drug Commercial name Polymer Solvents & other excipients Ref
Terbinafine
HPMC E-15
PEG 400
Ethanol
Purified water (132)
Poly (4-vinyl phenol) Ethyl acetate
Dibutyl phthalate
Triethyl citrate
Hydroxypropyl-β-cyclodextrin
Isopropyl alcohol
Acetone
Cellulose acetate
Ethyl cellulose (57)
Ketoconazole
Propylene glycol
PEG 400
Glycerin
Urea hydrogen peroxide
Ethanol
Thioglycolic acid
(133)
Polysilicone-8
Acrylates copolymer
Ethanol
Panthenol
Tocopheryl acetate
Phytantriol
Butylene glycol
Benzophenone-3
Calcium chloride
Fragrance
(134)
Miconazole Nitrocellulose
Propylene glycol
Urea hydrogen peroxide
Salicylic acid
Ethanol
Transcutol® P
Ethanol
(135)
EV-086K
Eudragit® E100 (111)
Amorolfine
Loceryl®
Metacrylic acid
copolymer
Glycerol triacetate
Butyl acetate
Ethyl acetate
Ethanol
(110)
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Ciclopiroxolamine belongs to the group of hydroxyl-pyridone derivatives, and it has been
used as a nail lacquer to treat onychomycosis since 1990. It exerts its antifungal activity by
chelating trivalent cations, like Fe3+ and Al3+, compromising fungal metal-dependent enzymes
and reducing the fungus nutrient intake. Ciclopirox is active against dermatophytes
(Trichophyton spp., Microsporum spp., Epidermophyton floccosum), yeasts (Candida spp.,
Malassezia furfur, Cryptococcus neoformans, Sacchromyces cerevisiae), molds (Aspergillus
spp., Scopulariopsis brevicaulis, Fusarium solani) and some bacteria, which is advantageous
in cases of mixed infection. It is also reported to have some anti-inflammatory activity by
inhibiting the local production of prostaglandins and leukotrienes. The nail lacquer available
in the market has a concentration of 8% ciclopirox, which increases to 35% after application
and evaporation of the volatile solvents. Studies show that it can exceed MIC for the three
most important onychomycosis pathogens–T.rubrum, T. mentagrophytes, and Candida
albicans. Common posology is once daily application for 6-12 months over the clean nail
plate and slightly over the surrounding skin. However, there are trials stating that once the
weekly application is effective. Clinical trials have reported complete cure in 29-36% of
patients. Ciclopirox is available in both Europe and United Stated of American: Mycoster®,
Niogermos®, Batrafen®, Kitonail®, Onytec®, Stieprox®, Loprox®, Penlac®, Ciclopoli®
and RejuveNail® (77,83,88–94).
Besides these main drugs, others have been recently studied as well as specific excipients as
follows.
Monti et al. (131) purposed to evaluate the water-soluble film forming agent hydroxypropyl
chitosan included in an experimental nail lacquer (P-3051) containing ciclopirox.
Hydroxypropyl chitosan is a water-soluble derivative of chitosan. Chitosans are
polysaccharides derived from chitin and natural components of the exoskeleton of crustaceans
being widely employed in medicine for their wound healing, bacteriostatic, skin moisturizing,
and protecting properties. In particular, hydroxypropyl chitosan was chosen considering its
favorable properties, such as high water solubility; high plasticity; affinity to keratin; wound-
healing activity; high compatibility with human tissues, etc. P-3051 was composed by 1%
hydroxypropyl chitosan, 1% cetostearyl alcohol, 73% ethyl alcohol (95°), 4% ethyl acetate
and 13% purified water. This formulation was compared with a commercial brand (Penlac™)
constituted by 8% ciclopirox, ethyl acetate, isopropyl alcohol, butyl monoester of poly
(methylvinyl ether/maleic acid) in isopropyl alcohol. Bovine hoof membranes were used as
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the human nail plate model. Drug concentrations were determined by HPLC (high-
performance liquid chromatography). Regarding lag times, the respective values obtained
were 3.36±0.46 h for P-3051 vs. 12.48±1.31 for Penlac™. The percent of permeated drug
(Q%30h) was also significantly different for the two formulations: 2.58% for P-3051 vs.
1.06% for Penlac™. In fact, a faster drug penetration time might allow the drug to permeate
the nail before the hydro soluble film is degraded. Greater efficiency of P-3051 could be
attributed to a affinity of hydroxypropyl chitosan to the nail matrix, resulting in an intimate
contact and strong adhesion of the lacquer to keratin substrate (131).
An in vitro study with the formulation previously developed by Monti et al (131) reported an
achievement of 13% complete cure rate, which was quite low. It was concluded that
ciclopirox formulated in the new hydrolacquer technology is more active and better tolerated
than the reference ciclopirox nail lacquer for the long-term treatment of onychomycosis. In
addition, it is much easier to apply without needing any bothersome removal procedures
(136).
In 2009, Monti et al. compared the transungual permeation of ciclopirox with amorolfine
vehiculated in the same hydroxypropyl chitosan-based lacquer and a water insoluble
reference (Loceryl® containing Eudragit® RL100, triacetine, butyl acetate, ethyl acetate,
ethyl alcohol and drug). The study was performed on bovine hoof slices, and drug
concentration was determined by HPLC. Amorolfine experimental lacquer showed higher
permeation than the commercial water-insoluble option. Also, ciclopirox lacquers showed a
better performance than amorolfine lacquers (114).
Later in 2012, a clinical trial with these formulations was conducted (56). The results
supported in vitro data except from day 15 to day 25 in which the nail concentration of these
drugs decreased. The authors hypothesized that the steady state had not been reached yet.
Moreover, in vitro studies do not take into account all the environmental interactions that
occur under in vivo conditions where both fingers and toenails are constantly exposed to
tissues, liquids, and blood circulation, resulting in some drug loss. This may be a disadvantage
for water soluble formulations since the contact with water can lead to a loss of medication;
however, it can be countered by a more frequent application (56).
Chouhan et al. (57) studied the influence of a permeation enhancer hydroxypropyl-β-
cyclodextrin in a terbinafine nail lacquer formulation. This formulation was composed of
cellulose acetate and ethyl cellulose as film forming polymers, triethyl citrate as a plasticizer,
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and isopropyl alcohol and acetone as solvents. Formulations containing this enhancer
demonstrated a higher flux than the control formulation in in vitro studies. The lacquer
containing 10% (w/v) hydroxypropyl-β-cyclodextrin showed maximum flux of 4.586± 0.08
µg/mL/cm2 as compared to the control flux of 0.868 ± 0.06 µg/mL/cm2, demonstrating its
ability to enhance the transungual permeation of poorly soluble drugs (57).
A ketoconazole nail lacquer for onychomycosis treatment was also described by Kiran et al.
in 2010 (122). The studied formulations contained ketoconazole, propylene glycol, glycerin,
ethanol, polyethylene glycol 400, thioglycolic acid and urea solution in H2O2. Several factors
were evaluated including non-volatile content, drying time, smoothness of flow, gloss and
permeation studies in human nail plates. Urea hydrogen peroxide enhanced hydration state
and thioglycolic acid cleaved keratin bonds, allowing better penetration results which could
be promising upon formulation optimization (133).
In 2011, a novel lacquer formulation for the transungual delivery of ketoconazole was
reported by Hafeez et al. The vehicle used had an anhydrous/alcohol composition with a dual
acrylate-silicone hybrid copolymer system that offered film forming and occlusion properties
due to synergistic plasticizing components (134). It contained ethanol, polysilicone-8,
panthenol, acrylates copolymer, tocopheryl acetate, phytantriol, butylene glycol,
benzophenone-3, calcium chloride and fragrance, as well as radiolabeled [1-14C]-
Ketoconazole. This test formulation was compared with a commercial ketoconazole cream.
The study was conducted on human nails placed in a diffusion cell simulating physiological
conditions. The formulations were then administered once daily for 7 days. Sampling was
made by drilling and radioactivity was measured from the samples obtained. Following the 7-
day exposure, the ketoconazole content measured in the ventral/intermediate layers was 0.81
± 0.39 µg/mg or 535 ± 260 µg/cm3 for the lacquer formulation and 0.09 ± 0.05 µg/mg or 53 ±
29 µg/cm3 for the control formulation. The drug concentration attained in the nail was
approximately 2,140 times higher than the MIC for common dermatophytes, exceeding the
MIC for most common onychomycosis pathogens. According to these results, this lacquer
formulation can be a potential effective topical treatment for onychomycosis (134).
Hui et al. (69) assessed the enhancing properties of 2-n-nonyl-1,3-dioxolane in a lacquer
formulation (EcoNail™) to increase the permeation of econazole. The test formulation
contained 5% (w/w) econazole, Eudragit® RL/PO, ethanol and 18% (w/w) 2-n-nonyl-1,3-
dioxolane. The assay was performed on healthy human nail plates collected from cadavers
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and using a diffusion cell. An aliquot of all tested formulations was applied on the nails twice
daily for 14 days, washing the nail plates between applications. After incubation, nail samples
were obtained with a drill and radioactivity of labeled compounds was measured. The
concentration and flux of econazole into the deep layer of the human nail in the test group
was about 6.3-fold and 7.5- fold higher than the control group, respectively. Econazole
concentration was about 15,000 times the MIC for most dermatophytes species and 150 times
that for most molds. On the contrary, dioxolane did not penetrate to deeper layers of the nail.
These authors hypothesized that besides facilitating diffusion of the drug, dioxolane also
functioned as an adhesion promoter and plasticizer for the film-forming polymer, softening
the Eudragit film in the lacquer. These results suggest that 2-n-nonyl-1,3-dioxolane-enhanced
econazole lacquer has the potential to be an effective topical treatment for onychomycosis
(69).
The same author performed another study (78) in which reported the nail penetration of
tavaborole (AN2690) from different vehicles and compared with ciclopirox. Four
formulations, all containing 10% (w/w) AN2690 were compared for their ability to deliver
AN2690 to the deep layers of the nail plate and into the nail bed. The composition (w/w) of
these different vehicles was: formulation A: 70% ethanol and 20% poly (vinyl methyl ether alt
maleic acid monobutylester), a polymer that forms a water insoluble film, very durable and
resistant to damage; formulation B: 56% ethanol, 14% water, 15% poly (2-hydroxyethyl
methacrylate) and 5% dibutyl sebacate, forming a water soluble film; formulation C: 55%
ethanol, 15% ethyl acetate, 15% poly (vinylacetate) and 5% dibutyl sebacate, forming a water
insoluble film that can be removed by peeling or scratching the surface; formulation D: 20%
propylene glycol and 70% ethanol (only solvents). Aliquots of those formulations were
applied on human nail plates once daily for 14 days. The ventral/intermediate nail samples
were collected at the end of the 14th day dose period, stored at 4°C and analyzed for drug by
LC/MS/MS. Considering that any formulation showed a distinct advantage over the others,
the simplest formulation (D) was chosen for further development (78).
Baran et al. (68) evaluated the efficacy of a fluconazole-urea nail lacquer in onychomycosis
treatment of 13 patients. The lacquer was composed of 1% fluconazole, 20% urea,
polyvinylpyrrolidone k25, glycerol triacetate, docusate sodium, ethanol and demineralized
water. After 12-18 months’ treatment, about 90% of patients had positive results regarding
clinical and mycological cure (68).
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A terbinafine bilayered nail lacquer for onychomycosis was developed by Shivakumar et
al. in 2010. Since aqueous-based nail lacquers promote nail hydration and drug diffusion
through the nail but lack durability, the authors studied an underlying drug-loaded hydrophilic
lacquer with an overlying water resistant film. The hydrophilic nail lacquer was composed of
5% (w/v) terbinafine, 6% (w/v) hydroxypropyl chitosan E-15 (water soluble polymer), 10%
(v/v) PEG 400 (penetration enhancer, plasticizer and humectant), 60% (v/v) ethanol and 19%
purified water. Formulation controls as a hydrophilic nail lacquer of E-15 containing
terbinafine but devoid of PEG 400 and a placebo (formulation without the active drg)
hydrophilic lacquer of hydroxypropyl chitosan E-15 containing PEG 400 were similarly
prepared for comparison. Briefly, terbinafine was dissolved in a mixture of water and ethanol
(pH 3.0) by a bath sonicator. Hydroxypropyl chitosan E-15 was soaked overnight in the
hydroalcoholic mixture (pH 3.0) and sonicated to ensure complete polymer dissolution. The
two hydroalcoholic solutions were mixed and stirred to obtain a clear homogeneous solution
to which PEG 400 was added. The hydrophobic nail lacquer was prepared by dissolving poly
(4-vinyl phenol) in ethyl acetate at 10% (w/v). Dibutyl phthalate was used as a plasticizer in
the lacquer at 4% (v/v). The pH, viscosity and drying time of the hydrophilic nail lacquers
were found to be around 4.0, 500 cps, and 300±75s, respectively. In vitro drug permeation
studies were performed with cadaver nails. Although therapeutic concentration values were
reached in in vitro studies, a clinical study on infected patients should be performed to
ascertain clear results (132).
Vipin et al. (135) presented in 2014 a miconazole nail lacquer. This formulation included a
film former (nitrocellulose), permeation enhancers (urea in hydrogen peroxide and propylene
glycol), a keratolytic agent (salicylic acid) and an antifungal agent (miconazole nitrate) in
ethanol. Several formulations were tested regarding nonvolatile content, gloss, smoothness to
flow, drug release (bovine hoof model), drug content (UV spectrophotometry) and antifungal
activity. Among ten formulations, the nail lacquer prepared with 2% drug, 3% nitrocellulose,
0.5% ethyl cellulose, 20% salicylic acid, 5% propylene glycol and 5% urea in H2O2 exhibited
the best results, being a promising formulation for the treatment of Candida albicans (135).
Naumann et al. (111) studied the controlled delivery of EV-086K nail lacquer through the
nail plate. 5% Eudragit® E100, dimethylaminoethyl methacrylate, butyl methacrylate and
methyl methacrylate were mixed with 85% ethanol and stirred until the polymer was
completely dissolved. 5% EV-086K and 5% Transcutol® P were stirred in a separate amber
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glass until a clear solution was obtained. Then, the polymer–ethanol mixture was added.
Bovine hoof slices, equine hood slices, and human nails were employed for delivery studies.
This nail lacquer penetrated into the nail at higher concentrations than other studied
formulations. However, it did not reach the acceptor compartment (111).
5. Polyurethane a potential excipient in therapeutic nail lacquer
The polyurethanes (PU) are among one of the most important class of specialty polymers
(137)). They are obtained by the reaction between isocyanate and hydroxyl compounds (138).
The polyurethane production was developed by Otto Bayer and co-workers in 1937 (139) for
the polyaddition reaction. Since then the polyurethanes have found many different
applications and the polyurethanes market has been growing uninterruptedly (140).
Polyurethanes are polymers containing urethane linkages (–NHCOO–) in the main polymer
chain. They can be classified into the following major groups: flexible foams, rigid foams,
elastomers, fibers, molding compositions, surface coatings and adhesives (137).
5.1 Synthesis of polyurethane
Polyurethanes are synthesized from three components, namely a polyol (polyglycol), a
diisocyanate and a chain extender (diol) as shown in Figure 1.6.
Figure 1. 6. Scheme of Synthesis of polyurethane adapted from (141).
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They present a segmented architecture, where in the diisocyanate and the chain extender (diol
of low molecular weight) form the hard segments of the polymer chain and the polyol of high
molecular weight (interception sections) form the soft segments as represent the Figure 1.7.
Figure 1. 7. Architecture of the chain of polyurethane. Adapted from (142)
The hard and soft segments of the polyurethane are responsible for the wide range of
elastomeric properties presented by this polymer. The soft segments, owing to their low glass
transition temperatures are responsible for the elasticity of the polyurethane. However, the
hard segments make the polymer stiffer due to the presence of inter-chain hydrogen bonds
between the hard segment in the polymer. The hydrogen bonding causes the hard segments to
pack themselves into crystalline domains which function as physical crosslinks between the
polymer chains.
For the producing a material with a broad range of mechanical properties which is both elastic
and strong, the architecture of the polymer is modulated by variations such as:
• The ratio of the hard segment to the diol.
• The choice of the hard segment and soft segment components (139).
5.2 Monomers for the synthesis of polyurethanes
Polyglycols of high molecular weight
Convenient long chain polyglycols such as polyethylene oxide diols, linear or ramified, and
polyester diols are used as monomers for the synthesis of PU (137,138,139) (Table 1.7).
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Table 1. 7. Polyglycols employing in synthesis of polyurethanes.
Polyglycols Chemical structure
Poly (tetramethylene oxide)
Poly (propylene glycol)
Poly (ethylene glycol)
Poly (dimethyl siloxane)
Poly ε-caprolactone
Diisocyanates
In order to obtain non-toxic polyurethanes, the diisocyanates (138,139), employing are
representing in Table 1.8.
Table 1. 8. Name, toxicity and chemical structure of diisocyanates.
Diisocyanates LD50(mg/Kg) Chemical structure
HMDI 9 900
IPDI 1000
LDI Value no relevant
HDI Value no relevant
MDI 2 200
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Chain extenders
Carbohydrates are frequently employed as chain extenders in the architecture of the polymer.
Some examples of the synthesis of polyurethanes from a renewable source (143) are shown in
Table 1.9.
Table 1. 9. Carbohydrates employed as chain extender in the synthesis of polyurethanes.
Chain extender Chemical structure
Sucrose
Fructose
Glucose
Galactose
Lactose
Maltose
Sorbitol
Isosorbide
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Catalysts
Currently, the flexible polyurethanes are made with the aid of at least one catalyst (139). They
ensure the completion of the reaction in the polyurethane. Catalysts controlling the reaction
time and for defining polymer architecture that influences the ultimate mechanical properties.
Specifically, it is the catalyst’s activity and selectivity towards each of the many reactions
occurring in the formation of polyurethanes that determine the structure of the resulting
material. The amine and organometallic are among the most used catalysts employing in the
synthesis of polyurethane, which showed in Table 1.10.
Table 1. 10. The most common catalyst in synthesis of polyurethane.
Catalyst Structure Class
Dibutyltin dilaurate
Organometallic
Tin(II) 2- ethylhexanoate Sn
Organometallic
Bismuth 2- ethylhexanoate
Bi
Organometallic
DMDEE
amine
DABCO
Amine
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5.3 Methods for synthesis of polyurethanes
The polyurethanes are synthesized by three methods:
• the one-shot method,
• the prepolymer method,
• the quasi-prepolymer method (144).
The prepolymer method and the quasi-prepolymer method correspond to processes known as
‘two-step methods’ or ‘two-step polymerization’.
The one-shot method is a process that takes place in one step. All the ingredients are mixed
simultaneously, and the resulting mixture is allowed polymerize.
In the prepolymer method, the macroglycol is pre-reacted with an excess of poly-isocyanate.
This prepolymer is then mixed with the rest of the ingredients during processing.
In the quasi-prepolymer method, a part of the diol is mixed with the isocyanate and the rest
of the polymer and the other constituents are mixed as a second phase.
In the pre-polymer process, the free content of isocyanate result of the reaction between the
diisocyanate and polyol is about 1-12%. On the other hand the quasi-prepolymer present
generally more that 25% of free isocyanate, in this process the NCO/OH ratio is 4:1, to yield a
low viscosity system consisting of a pre-polymer dissolved in excess of isocyanate (137).
Advantages of quasi-prepolymer.
- Allows maintain better control the structure and properties of the final polymer. Through a
simple change of chain extender, it is possible to obtain polyurethanes with different
properties from the same pre- polymer.
- The pre- polymers are typically liquids, which facilitates handling on an industrial scale.
- When compared with monomeric isocyanate systems, the prepolymers have a lower vapor
pressure and consequently lower toxicity.
- The viscosity of the prepolymers is higher than that of the monomeric isocyanates, which
allows a better homogenization when mixed with the polyol and better control of the
rheological properties when trying to lose fluidity.
- Allows better temperature control because the resulting heat bond formation is released in
two stages (145).
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5.4 Synthesis of polyurethanes from carbohydrates
Kennedy et al, 1986 describe preparing polyurethanes from carbohydrates such as pectin and
sucrose, one amine was employing as a catalyst. The syntheses were developed from
polyisocyanates at temperatures above 60 °C, yielding highly crystalline polymers (146).
Polyurethanes from prepolymers were obtained using different amounts of milliequivalents
sucrose, glucose, and fructose which reacted with MDI and PEG. In the polyurethanes
obtained was observed that the increasing of the content of saccharides influence in values of
glass transition temperature (Tg), Young's modulus and tension stress. The synthesis of the
prepolymer and polyurethane were developed at 25 ° C without solvent (147).
They have been reported polyurethanes employing glycosylamines and glucosamines
modifications. Only modified near the anomeric hydroxyl group, being the most reactive.
Hexamethylene diisocyanate (HDI) and 2,5 dianhydro -2,5 -dideoxy - L- iditol - diisocyanate
is used. The polymers were soluble in dimethylformamide, and their molecular weights were
between 53 655-75 300, (148).
In 2001, were proposed two polyurethanes obtained from partially protected carbohydrates.
They developed reactions of HDI with methyl 2,6-di- O- pyranosyl - α - D - glucopyranose
and methyl 4,6-O- benzylene - α -D - glucopyranose, in the ratio 1: 1, using 1,4- diazabicyclo
catalyst (2,2,2) octane. Tetrahydrofuran was used as a solvent in the reaction, obtaining
polyurethanes with values of Tg between 147-150ºC (149).
According to polyaddition catalytic process, polyurethanes using monomers D- Sorbitol, L -
iditol and D - mannitol were synthesized. The method was developed at 80 oC, with a stream
of nitrogen (143).
In 2005 was synthesized a polyurethane from sucrose and PEG 600 and 1000. The polymer
was not toxic (151). According to acute oral toxicity test, the administration of the
polyurethane did not present any clinical signs in any of the animals treated or damages in
the organs analyzed, which suggest that the polyurethane was a safe product (150).
In 2013 Aurelie Boyer and collaborates, synthesise a linear polyurethane from
transesterification of sucrose and α-d glucopyranoside. In the research was employing dibutyl
tin dilaurate as catalyst (151).
The synthesis of the linear polymer has many applications in research of Vicent Besse et al,
the aim of the work was obtaining a polyurethane with higher chemical and hydrolysis
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resistance. The polyhydroxyurethane was obtained by reaction of isosorbide, hydroxylated
polybutadiene and methylene diphenyl-4,4 diisocyanate (152).
Another example of the synthesis of polyurethanes from carbohydrate was the polyurethane
synthesized from the reaction of hexamethylene diisocyanate and various ratios of isosorbide
to poly(tetra methylene glycol) by one-shot bulk polymerization without a catalyst. The rigid
diols impart biocompatible and bioactive properties to thermoplastic polyurethane elastomers.
The degradation test showed loss of mass in the polyurethane from 4–9% after 8 weeks. In the
case of polymers with lowest isosorbide content, the weight loss was in a short time compared
with polyurethane that present highest isosorbide content (153).
The L-lactide is another carbohydrate to permit synthesized biodegradable crosslinked
polyurethane using polyethylene glycol and hexamethylene diisocyanate (HDI). In the
reaction, the iron acetylacetonate was using as catalyst (154).
5.5 Application of polyurethane in biomedical industry
The polyurethanes are copolymers which have been used for biomedical applications for over
three decades.
These desirable properties attract the attention of developers of biomedical devices. In 1958,
polyurethane materials were first introduced in biomedical applications; Pangman described a
composite breast prosthesis covered with a polyester-urethane foam (155). In the same year,
Mandrino and Salvatore also used a rigid polyester-urethane foam called OstamerTM for in
situ bone fixation (156).
Three years later, the application of polyester-urethane Polyurethane Estane® VC was
proposed by Dreyer et al to be used as components for heart valves and chambers, and aortic
grafts (157). In the mid-1960s, Cordis Corp. started to commercialize polyester-urethane
diagnostic catheters (137).
In 1954, textile chemistry at DuPont developed Lycra® spandex as a high-performance
alternative to natural rubber in elastic thread. It was first introduced as a biomaterial in 1967
by Boretos and Pierce who obtained the polymer in solution directly from the DuPont
spinning line that produced Lycra spandex yarn. This material was first used as the
elastomeric components of a cardiac assist pump and its arterial cannula (156).
The years 1970-1972, were a time of designed of polyurethane specifically commercialized
for medical use; Avcothane®-51TM, a polyurethane/silicone hybrid, Biomer®TM, a version
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of Lycra® T-126. Avcothane® and Biomer® were regarded as the first ‘real’ biomedical
polyurethanes, where some of them (156). Avcothane® was used clinically in the first intra-
aortic balloon pump (IAB), starting in about 1971, and is still in clinical use today in
IABs(90). Biomer®TM components were used in the ‘Jarvik Heart’ in 1982, the first artificial
heart used for implantation (137).
In the Figure 1.8 showed the application of polyurethanes in biomedical. These polymers
have been applied in some biomedical tissue engineering areas such as pacemaker lead
insulators, heart valves, vascular prostheses, breast implants, gastric bubbles, drug release
carriers, etc.
Figure 1. 8. Biomedical application of polyurethanes.
5.6 Application of polyurethane in drug delivery system.
The polyurethane system is being used for sustained and controlled delivery of various dosage
forms. These systems are based on a physical combination of the drug with polymers and
kinetics of drug release is generally controlled by the diffusion phenomena through the
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polymer (159). Some examples of polyurethane with application for controlled release, are
described below:
Controlled delivery System employing polyurethane and nystatin was development in 2001
by Michaela Mandru, et al (160). Sustained release of nystatin from polyurethane membrane
for biomedical application. The system was prepared by a mixture of a different amount of
polyurethane and nystatin. The final system consists of a membrane of polymers with nystatin
obtained by phase inversion methods. The mechanism for release of nystatin was according to
fickian diffusion.
Lijuan Zhou et, al in 2011, synthesized a pH-sensitive polyurethanes. For the synthesis of
polymers was used LDI, PCL-Hyd-PEG- Hyd-PCL, and tripeptide chain extender. The
reaction took place in two step. The pH-sensitive polymers were employing for anticancer
drug delivery. The polyurethane was degrading at pH 5-6, and release the antitumoral drug in
tumor site (161).
In other hand systems consisting of albumin nanoparticles were dispersed into a carboxylated
polyurethane. The release of drug was by diffusion phenomen, which observed in Figure 1.9,
(162).
Figure 1. 9. Release of nanoparticles from polyurethane matrix. Adapted from (162)
In 2006 the polyurethane was evaluated as theophylline delivery matrices. The polymers
were synthesized from aliphatic diisocyanate (Lysine methyl ester diisocyanate), poly ε
(caprolactone) and 1,4 butanediamine. The release of drug from the polyurethane matrix was
according to fickian diffusion model. The release of theophylline was faster with the increase
of the hydrophilicity of the polyurethane (163).
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In 2008 Andrea Hafeman et al, showed a polyurethane for release a growing factor (I-PDGF)
for tissue repair and regeneration. The polyurethane was prepared by the one-shot foaming
process. The controlled release system formulated, described a two-stage release profile,
characterized by a 75% burst release within the first 24h and slower release thereafter. By 21
days 89% of the (I-PPGF) has eluted from the matrix of polyurethane (159).
Chlorhexidine diacetate polyurethane controlled delivery system with the employ of
polyurethane. In this work, the drug loaded to the film of polyurethane was released for 11
days. The kinetic of the system was the zero-order (164).
The polyurethane was used in the blend of chitosan microspheres. The system obtained
permitted to release two cardiovascular drugs: isoxuprine hydrochloride and calcium
dobesilate. The microsphere with chitosan and polyurethane were prepared by emulsion
cross-linking method. The drug release from the microsphere followed the fickian to non
fickian diffusion mechanism (165).
The matrix of poly (ester ether urethane)s with rifampicim were prepared to employ different
concentration of urethane groups. The release of drugs was higher than the minimum
inhibitory concentration of rifampicim for gram-positive bacteria (166).
Another example of the release of drug from polyurethane was the heparin. In this work, the
heparin was blended with the polymer. The quantitative of heparin released from the system
was controlled by the thickness of polyurethane films (167).
The polyurethane was synthesized with vancomycin. The medical device permits inhibition of
infection of the bone wound in a rat femoral segmental defect model. The antibiotic
quantitative released after 8 weeks exceeding the minimal concentration for bactericide
action. The system was promisor for the treatment of osteomyelitis (168).
In 2008, the 7-ter-butyldimethylsilyl-10-hydroxyl-camptothecin was employed in a
formulation with polyurethane. This drug formulation was studied for the treatment of cancer.
The drug was covalently incorporated into the delivery system and released 0,1% of the total
drug load to the polymer in 65 days (169).
Polyurethanes have a great application for controlled release of the drug. The use of such
compounds for the release of antifungal has not been explored so far. Thus, the use of
polyurethane for the active principle release against onychomycosis is still a challenge.
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Chapter 2: Synthesis and Characterization of Polyurethanes
employing Carbohydrates
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1.Introduction
The covering of drugs with biodegradable polymers is one of the processes for producing
sustained and controllable drug release systems, also preventing the degradation of the drugs
(1). Film coating has been successfully utilized to control the release of active ingredients,
prevent interaction between ingredients and external medium and increase the strength of the
dosage form by maintaining the product integrity during storage (2).
Biomaterials in drug delivery systems have an enormous impact on human health care (3).
During the last years, the synthesis of new polymers for this application has increased
markedly (4). One of the polymers that have recognized application for coatings in the
pharmaceutical industry is the polyurethane (1). Polyurethanes have generated large interest
among the research community because they offer the greatest versatility in compositions and
properties of any other family of polymers. The use of elastomeric polyurethanes as
implantable medical devices have demonstrated a combination of toughness, durability,
biocompatibility and biostability not achieved by any other available material (3).
The PUs are composed of repeating units of monomers bonded by the urethane group (4,5).
Their versatility arises from the possibility of producing tailored PU and PUU. These
materials are obtained by the addition of different chain extenders or by varying the pre-
polymers’ composition using different polyol structure and functionality, and also changing
the NCO content (6). The polyurethane has been applied in the medical and pharmaceutical
industries since 1970 as a biomaterial in a variety of situations, e.g. prosthesis (7,8),
microspheres (9), apposite (10,11), patches (12) and others (13–15). The PUs are
conventionally prepared by a so-called one-shot process (16,17) or by a prepolymer mixing
process (18–23). In these processes, the ionic centers are incorporated as chain extenders and
located among the hard segments. Consequently, the mobility of the ionic segments is very
much limited by the rigidity of the hard segments, and the ionic groups are not well exposed
to the particle surfaces, requiring more ionic groups to produce a stable dispersion (15,24).
The biocompatibility of the polyurethanes becomes an attractive alternative for application in
the pharmaceutical industry in therapeutic nail lacquer formulation (25).
In this study, we synthesized, five different quasi-prepolymer polyurethanes using PEG
(Mw1500) and pMDI. The quasi prepolymers were characterized by viscosity test, the content
of NCO and FTIR. Later was select one quasi pre-polymer to synthesize different
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polyurethanes employing carbohydrates as extensor monomers. Finally, the polyurethanes
were characterized by FTIR, (1H and 13C) NMR spectroscopy, DSC and cytotoxicity test.
2. Materials and methods
2.1 Materials
Polyethylene glycol (PEG) 1500, were purchased from Basf (Germany), sucrose, glucose,
fructose, isopropyl myristate, 2-methyl-2,4-pentanediol, triethanolamine were obtained from
Merck (Germany), DMSO, THF, polyethylene glycol 300, glycerin was obtained from
Sigma-Aldrich (Germany), ethanol was purchased from Across (France), Transcutol®GC and
Labrasol® were obtained from Gattefossé (France). The acetone was acquired from Panreac
(Spain). Polymeric 4,4-methylene diphenyl diisocyanate (pMDI) (Desmodur® 44V20L) was
supplied by Bayer Material Science. The molecular sieves 4 Å was acquired from Merck
(Germany).
2.2 Methods
2.2.1 Synthesis of the polyurethane quasi-prepolymers
The synthesis of quasi-prepolymers was carried out using the quasi-prepolymer method. The
quasi-prepolymers were prepared by reacting pMDI and PEG 1500 (purchased from Basf,
Germany) in a 50% w/v solution with DMSO and dried over molecular sieves 4 Å) in a glass
flask of 500 mL, under a dry nitrogen atmosphere. An inert atmosphere was used to avoid the
ingress of moisture and the consequent formation of urea linkages during synthesis. The
reaction was developed with mechanical stirring (400 rpm) at 40ºC and with drop-by-drop
addition of polyol for 8h. The syntheses of polyurethane quasi-prepolymers were done in
triplicate. Further syntheses were carried out under identical conditions in the presence of 0.5
mL of the catalyst triethanolamine, which was added to each polyol solution that later reacted
with the isocyanate (8,23). The ratios of monomers employing for obtained the quasi-
prepolymer polyurethanes were presented in Table 2.1.
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Table 2. 1. Relation of monomers for synthesis of quasi-prepolymers.
Pre-6 Pre -8 Pre-10 Pre-12 Pre-14
m (pMDI) (g) 100 100 100 100 100
m(PEG 1500)(g) 96 69 55 46 39
NCO/OH 1.1 1.4 1.8 2.2 2.6
2.2.2 Synthesis of polyurethanes
The synthesis of polyurethane was carried out in a reactor of 250 mL with a quasi-
prepolymer polyurethane selected. Later amount of carbohydrate (glucose, fructose or
sucrose) dissolved in 10 mL of DMSO was added by dropping according to the relation
presented in Table 2.2. The mixture was maintained under 400 rpm agitation for 1 hour, with
a nitrogen stream. PUS was the polyurethane obtained employing sucrose as extender, PUF
was the polyurethane obtained employing fructose as extender and PUG was the
polyurethane obtained employing glucose as extender.
Table 2. 2. Relation of monomers for synthesis of polyurethane.
PU NCO/OH Carbohydrate
PUS 14/4 1 Sucrose
PUS 14/6 0.75 Sucrose
PUS 14/8 0.5 Sucrose
PUF 14/4 1 Fructose
PUF 14/6 0.75 Fructose
PUF 14/8 0.5 Fructose
PUG 14/4 1 Glucose
PUG 14/6 0.75 Glucose
PUG 14/8 0.5 Glucose
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2.3 Characterization of polyurethane quasi-prepolymers
2.3.1 Determination of viscosity
The viscosity of the quasi-prepolymer polyurethanes was determined by parallel plate
rheometer in a shear stress-controlled ICI, London, LTD rheometer, using parallel plates
(upper plate diameter ¼ 20 mm); the gap selected was 0.4 mm. The viscosity was measured at
25 0C and a frequency of 20 Hz.
2.3.2 Free isocyanate content
The NCO content in the quasi-prepolymer polyurethanes was determined by back titration of
excess N,N-dibutylamine with standard HCl, described in research of Daniela da Silva et al
2008 (12).
2.3.3 FTIR
The FTIR spectra were obtained with a Nexus Thermo Nicolet spectrometer equipped with
attenuated total reflectance (ATR) device. 128 scans with a spectral resolution of 4 cm-1 were
averaged to give the specimen spectrum from 4000 cm-1 to 600 cm-1. All FTIR spectra were
recorded at room temperature.
2.4 Polyurethane characterization
2.4.1 Solubility studies for polyurethanes
Polyurethane was added to different vehicles such as water, water-ethanol (1:1), ethanol,
Transcutol®GC, isopropyl myristate, 2-methyl-2,4-pentanediol, glycerin, PEG 300, acetone,
Labrasol® and DMSO.
2.4.2 FTIR
The FTIR spectra were obtained according description in section 2.3.3 in the present chapter.
2.4.3 NMR
1H NMR and 13C NMR spectra in the liquid state were obtained using a BRUKER 300 MHz
spectrometer. Samples were dissolved in CDCl3, at room temperature (the spectra were
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referred internally using HDMSO, H 0.058 and C 1.9 relative to TMS. The measurement
was done using 10 mg samples of samples.
2.4.4 Thermoanalytical measurements
Thermoanalytical measurements were performed with a Mettler DSC system (Mettler,
Switzerland) with a sample robot TS0801RO (Mettler, Switzerland). The sample and the
reference (air) were placed in hermetically sealed pans. A scan speed of 10 °C/ min was used,
from -100oC to 250oC. The weight of each sample were 5-6 mg. These values gave the best
compromise between resolution, temperature accuracy and attenuation.
2.4.5 Cytotoxicity assay
2.4.5.1 Preparation of sample
For this study, 350 mg of polyurethane in solid stated with a dimension of 0.5x 0.5cm was
tested.
2.4.5.2 Direct contact assay
The biocompatibility of the polymer was evaluated in vitro by direct contact with cells,
following the ISO 10993-5:2009 recommendation guidelines(26). Each material was added to
a 0.5 mL of HaCaT cell suspension [a spontaneously immortalized human keratinocyte cell
line (CLS, Germany)], in fresh culture medium [RPMI-1640® (Gibco, UK) medium
supplemented with 10% fetal bovine serum (FCS, Life Technologies, Inc., UK), penicillin
(100 IU/mL), and streptomycin (100 μg/mL)] (2.5 x 104 cells/mL), in sterile 24-well plates.
Glass slides without any formulation were used as control. The plates were incubated for 72 h
in a humidified atmosphere of 5% CO2 at 37 °C, without refreshing the culture medium. For
cell proliferation quantification, the general cell viability endpoint MTT reduction (3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was used (27,28). Accordingly,
the previous culture medium was removed and replaced with a fresh medium containing 0.5
mg/mL of MTT. The cells were further incubated for 3 h. In the plates containing the reduced
MTT, the medium was removed and the intracellular formazan crystals were solubilized and
extracted with dimethylsulfoxide. After 15 min, at room temperature, the absorbance was
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measured at 570 nm in a Microplate Reader (FLUOstar Omega, BMGLabtech, Germany).
The relative cell viability (%) compared to control cells was calculated using Equation 1:
(1)
The OD, is the optical density measurement. The data are expressed as the mean and the
respective standard deviation (mean ± SD) of 6 experiments. The statistical evaluation of data
was performed using one-way analysis of variance (ANOVA). The Tukey–Kramer multiple
comparison tests (GraphPad PRISM 5 software, USA), were used to compare the significance
of the difference between the groups; a p < 0.05 was accepted as statistically significant.
3. Results and discussion
3.1 Syntheses of polyurethane quasi-prepolymers
The chemical structures proposed for the polyurethane quasi-prepolymers are given in Figure
2.1. All the quasi-prepolymers were obtained in the liquid phase and presented an amber
color.
32
OO
OO NHC
ONH C
O
CH 2 CH 2
NCO
n
OCNCH 2 CH 2 NCO
NCO
n
CH 2 CH 2 NCO
NCO
n
OCN + OO
OO
32
HH
Figure 2. 1. Scheme of the quasi-prepolymer reaction.
3.2 Viscosity and NCO content of polyurethane quasi-prepolymers
The NCO content and viscosity of all polyurethane quasi-prepolymers synthesized are
presented in Table 2.3. The viscosity values of the samples decreased when the NCO content
increased. These results are in good agreement with the results of other authors. A.L. Daniel
pMDI PEG
Polyurethane quasi-prepolymer
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et al. 2007 (10), who comment that the decreasing of the NCO/OH molar ratio produced an
increase in the average molecular weight and viscosity of the quasi-prepolymers. Almost all
the quasi-prepolymers remained liquids for five months after the synthesis, maintaining
constant viscosity values. Hardening was only observed for the Pre-6 sample on the seventh
day after the synthesis. Table 2.3 shows a viscosity of 24 Pa.s for this quasi pre-polymer,
which might indicate that it possesses the highest molecular weight and the highest degree of
crosslinking than the rest of the quasi-prepolymers. The quasi-prepolymer 14 was the
polymers with the highest percent of isocyanate free.
Table 2. 3. Formulation, isocyanate content and viscosity of the polyurethane quasi pre-
polymers. (mean±SD, n=3).
Polyurethane
quasi-
prepolymer
m(pMDI)
g
m(PEG)
g
NCO/OH
m/m % NCO
free
Viscosity
(Pa.s)
Pre – 6 100 92 1.1 9 ±0.3 24 ± 0.1
Pre – 8 100 69 1.4 10 ±0.3 11 ± 0.1
Pre – 10 100 55 1.8 13 ±0.6 10 ± 0.1
Pre – 12 100 46 2.2 14±0.2 8 ± 0.2
Pre – 14 100 39 2.6 15±0.4 5 ± 0.1
3.3 FTIR analysis of polyurethane quasi pre-polymers
All quasi polyurethane pre-polymers show a similar FTIR spectrum. In Figure 2.2 and Figure
2.3 were presented the spectrum of quasi pre-polymers and the monomers employing in the
syntheses. The weak band to NH around 3000 cm-1 was not observed. The bands between
2980-2976 cm-1 corresponding to CH2 groups of PEG were observed. The strong band at
2265–2260 cm-1 confirms the existence of free NCO groups in the quasi-prepolymer
polyurethane (10,15). The band from 1716 to 1712 cm-1 was attributed to the stretching
vibration of C=O bond (13,15) characteristic of urethane group, due to the deformation of the
NH bond and C–N stretching vibration. The peak about 1600-1595 cm-1 corresponds to C=C
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stretching in the aromatic ring (6,8). Two peaks at 1012 and 1510 cm-1 arise from symmetric
and asymmetric stretching vibrations of N–C–N, respectively, corresponding to the reactions
of the NCO groups with the OH groups (22). This behavior has been reported previously for
polyurethane (10). The DMSO did not influence in the synthesis process of the quasi-
prepolymers.
Figure 2. 2. FTIR in ATR mode spectra of the Pre-6, Pre-8, Pre10 and Pre-12.
Figure 2. 3. FTIR in ATR mode spectra of the Pre-14, PEG 1500, DMSO and pMDI.
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3.4 Synthesis and characterization of polyurethanes
The polyurethane quasi-prepolymers hardening in contact with the chain extender in the few
minutes of the beginning of the syntheses, except in the synthesis were Pre-14 was employed.
These results were due to the high viscosity of the quasi-prepolymers and a low content of
free NCO, which causes immediate crosslinking.
The quasi-prepolymer 14 presents the highest content of isocyanate free (see Table 2.3), it
was selected for the syntheses of polyurethanes. Different content of sucrose, fructose or
glucose were incorporated, leading to ratios NCO/OH 1, 0.75 and 0.50.
3.4.1 Solubility of PU
The solubility of PUs was studied in different solvents. Only the PUS 14/4 present solubility
in DMSO, in the rest of solvent, the polymers not present solubility. The polymers: PUS
14/6, PUS 14/8, PUG 14/4, PUG 14/6, PUG 14/8, PUF 14/4, PUF 14/6 and PUF 14/8, not
present solubility in the solvent study. In the literature is mentioned that the amount of
primary alcohol reacts first that the second alcohol in the same molecular (29). According
this, one hypothetical and ideal structured for these polyurethanes are presented in Figure 2.4.
Figure 2. 4. Ideal structure for polyurethanes: a) PUS, b) PUF, c) PUG.
a)
b)
c)
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However, the resulted obtained indicated that not only one hydroxyl group reacted with the
quasi-prepolymer polyurethane. In the real structure of polyurethane, all the hydroxyl group
from sucrose, fructose and glucose reacted with quasi-prepolymer and formed a hydrophobic,
non- linear polyurethane, as presented in Figure 2.5. These type of polymers does not present
solubility properties in the usually solvent employed in pharmaceutical industry, such as
ethanol (30).
R: quasi- prepolymer polyurethane
Figure 2. 5. Structure of polyurethanes obtained, a) PUS, b)PUF, c) PUG.
3.4.2 FTIR analysis of polyurethanes
The characterization by FTIR was performed only for the PU obtained from sucrose.
Comparative analysis of the full spectra (Figure 2.6) shows a similarity between the
polyurethanes obtained. This evidence indicates that it is possible to achieve the polyurethane
in all cases.
a)
b)
c)
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Figure 2. 6. FTIR in ATR mode spectra of polyurethanes prepared with Pre-14.
The presence of a weak band to 3326 cm-1, corresponding to the stretching vibrations of NH
groups is observed in all the polyurethanes synthesized. The confirmation of the complete
polymerization and formation of all urethane groups is obtained by the disappearance of the
band to 2265 – 2260 cm-1 in the spectrum and the formation the band from 1716 to 1712 cm-1
attributed to the stretching vibration of C=O bond (24,31,32) characteristic of urethane group.
3.4.3 NMR analysis of polyurethane
3.4.3.1 1HNMR of polyurethane
The analysis by NMR only will be obtained for PU14/4 because the rest of polyurethanes
don’t present solubility in the deuterated solvents used.
For the interpretation of polyurethanes spectra, the signal corresponding to PEG 1500, pMDI
and sucrose were studied
The structure of PEG 1500 presented in Figure 2.7, shows four chemically different protons,
its 1H-NMR spectrum is presented in Annex 1. In the Table 2.4 are observed the chemical
shifts obtained for PEG 1500, as well as those found in the literature. However, in the
presented spectra due to the chemical environmental those chemical shifts which should
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present distinguished peaks appeared very closed to each other, this effect was corroborated
by some studies found in literature.
Figure 2. 7. Structure of PEG.
Table 2. 4. Chemical shifts of PEG 1500.
Type of H Experimental chemical
shifts CDCl3 (ppm)
Chemical shifts of
reference*
CDCl3 (ppm)
1 3.38 3.4
2 3.83 3.6
3 3.62 3.6
* (33)
Furthermore, (see Annex 1) were observed the sign of hydrogen corresponding to hydrophilic
group. These results are similar to report for PEG in the literature (34).
Similarly, to previous compound, the structure of pMDI (see Figure 2.8) presented three types
of chemically different protons, observed in Table 2.5. The 1H-NMR spectrum of pMDI was
presented in Annex 2.
Figure 2. 8. Structure of pMDI.
HOO
OOH
1 2 3 12
3
32
CH2 NN CC OO
HH
H H H H
HH
1
2
2 2
2
3
3
3
3
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The detected signals are summarized in Table 2.5. There is complete correspondence with
shifts reported in the scientific literature consulted.
Table 2. 5. Chemical shifts of pMDI.
Type of H Chemical shifts
CDCL3 (ppm)
Chemical shifts
reference*
CDCL3 (ppm)
1 3.87 3.9
2 7.08 7.1
3 6.87 7.0
* (10)
The sucrose was another monomer for analyses, the 1H NMR spectrum of sucrose was
presented in Annex 2. The structure of disaccharide was presented in Figure 2.9 and in the
Table 2.6 are showed the chemical shifts.
Figure 2. 9. Structure of Sucrose.
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Table 2. 6. Chemical shifts of Sucrose.
Type of H Chemical shifts
CDCL3 (ppm)
Chemical shifts
reference*
CDCL3 (ppm)
1,7,8 3.94 3.8-3.9
2,3,4,5,6 4.37-4.88 4.37-4.9
9 5.40 5.3
10,11,12,13 3.6-3.9 2.4-4
14,15,16 4.05-4.51 4.1-4.5
17,18,19 3.57-3.90 3.5-3-85
*(32,35)
All the spectroscopy information obtained from the monomers employ in the synthesis of
polyurethane, permitted confirm the structure of the final polymer. The 1H-NMR spectrum of
PUS 14/4 is presented in Figure 2.10.
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ppm 0.01.02.03.04.05.06.07.08.0
7,9
44
7,9
03
7,8
74
7,8
73
7,2
74
7,0
31
7,0
24
6,9
57
6,9
35
6,9
29
4,6
54
4,2
51
4,2
36
4,2
32
4,2
21
3,8
51
3,8
19
3,5
71
3,3
32
2,9
23
2,7
77
2,5
50
2,3
97
2,3
18
2,1
04
2,0
51
1,1
91
0,0
02
Figure 2. 10. Spectrum 1H-NMR of PUS 14/4.
The signals presented in the spectrum corroborated the structure of polyurethane. Considering
the small amounts of sucrose employed for the synthesis of the macromolecule, most signals
of sugar are lost in the spectrum. However, in the spectrum appear signs between 2-4 ppm
that indicated the presence of sucrose in the polyurethane.
In the aromatic polyurethane section (6.9-7.3 ppm) observed increased complexity due to the
loss of symmetry of the aromatic protons compared with pMDI. In this case, it was not
possible to assign the signals of each one of the hydrogens. The sign corresponded CH2 to
aromatic isocyanate, was identified at 3.85 ppm, agree with the identified in other research
(10). On the other hand, signals between 3.3 to 3.5 ppm helped to identify the presence of
PEG 1500 in the structure of the polyurethane.
The characteristic signals of the protons bonded to heteroatoms, between 7.9 – 8.0 ppm
correspond to the amine groups present in the compound (10,32). These signals are
imperative because it confirms the proposed reaction between hydroxyl groups (PEG 1500)
and isocyanate groups (pMDI) (Figure 2.10).
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3.4.3.2 13C NMR of polyurethanes
To support the above results, another experiment was done, 13C-NMR. The 13C-NMR
spectrum is presented in Figure 2.11, the signals which are in line with the proposed structure
of the polyurethane.
ppm 050100150
15
3,4
7
15
2,9
9
12
9,6
8
12
4,5
6
11
8,7
9
77
,43
77
,00
76
,58
63
,69
40
,77
37
,29
1,7
3
Figure 2. 11. Spectrum 13C-NMR of PUS 14/4
The signals corresponding to sucrose lost in the 13C-NMR spectrum of polyurethane situation
previously detected in the assay of 1H-NMR. The sign of 40.6–41.6 ppm corresponded to CH2
of pMDI (32). The signs of carbons corresponding to PEG were found in 65 ppm. The
aromatic area of pMDI were observed at 70.4 ppm and 80 ppm. The signs detected between
118-154 ppm corresponding to carbonyl of polyurethane (36).
3.5 DSC analysis of polyurethanes
The study by DSC of polyurethanes synthesized employing sucrose showed different values
of Tg. This behavior is entirely logical considering that small variations in the formulation of
a polymer can lead to substantial changes in its structure(37).
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As a result of the analysis of polyurethanes PUS 14/4, PUS14/6 and PUS14/8, the values of
the respective Tg are shown in Figure 2.12. It was found temperatures in the range between
24.00 to 34.49 oC for the three studied cases, that correspond with Tg.
Figure 2. 12. DSC thermograms of polyurethane PUS 14/4, PUS 14/6, PUS 14/8.
Tg values in this study are less than those reported by Ganta (38), which means that the
structure of the polyurethane contains predominant soft segments confering flexibility to the
polymer, explaining thus why the developed materials have the characteristics of elastomers.
3.6 Results of cytotoxicity assay
This test was carried out for PUS14/4 and control slide glass (Figure. 2.13). Significant
morphologic change was observed in the cells in contact with the polyurethane, in the studied
time periods, the cells studied present different appearance compared with the structure of
HaCaT.
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Figure 2. 13. HaCaT cells after 72h of proliferation under contact with polyurethane. A glass
slide (control), B PUS 14/4.
The cell viability of PUS 14/4 was 21±2% (p<0.001) thus this polymer is not biocompatible
and thus not allow cell growth.
The incompatibility of the polyurethane probably present relation to the concentration of
DMSO residual.(39)
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4. Conclusions
Five quasi-prepolymers with free isocyanate between 9-15% and viscosity between 24-5 Pa.s
were synthesized. All the polyurethane quasi-prepolymers were characterized by FTIR. From
the quasi-prepolymer 14, three polyurethanes with different content of sucrose were obtained
with high cross-linking bonds density. The polyurethanes were characterized by
Spectroscopic techniques (FTIR and NMR). The glass transition temperature of the
polyurethanes obtained was between 24-34 °C, similar to elastomers. However the
biocompatibility of the polyurethanes obtained was affected probably by the structure of
pMDI and the DMSO residual. These preliminary results in the synthesis of polyurethane
from carbohydrate led the study of another isocyanate that permit to obtain polyurethanes
with better solubility and biocompatible with HaCaT cells.
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Studies on the morphology, properties and biocompatibility of aliphatic diisocyanate-
polycarbonate polyurethanes. Polym Degrad Stab. 2015;122:153–60.
28. Pyun DG, Choi HJ, Yoon HS, Thambi T, Lee DS. Polyurethane foam containing
rhEGF as a dressing material for healing diabetic wounds: Synthesis, characterization,
in vitro and in vivo studies. Colloids Surfaces B Biointerfaces. Elsevier B.V.;
2015;135:699–706.
29. Ritter FO. Differentiation between primary, secondary and terciary alcohols. J Chem
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30. Hofer R, Clark J, Kraus G, Saling P. Sustainable solutions for modern economies.
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31. Pereira IHL, Ayres E, Patrício PS, Góes AM, Gomide VS, Junior EP, et al.
Photopolymerizable and injectable polyurethanes for biomedical applications:
Synthesis and biocompatibility. Acta Biomater. 2010;6(8):3056–66.
32. Kizuka K, Inoue S. Synthesis and Properties of Polyurethane Elastomers Containing
Sucrose as a. 2015;(October):103–12.
33. Wacker BK, Scott EA, Kaneda MM, Alford SK, Elbert DL. Delivery of Sphingosine 1-
phosphate from poly(ethylene glycol) hydrogels. Biomacromolecules. 2006;7(4):1335–
43.
34. Garcia-Fuentes M, Torres D, Martín-Pastor M, Alonso MJ. Application of NMR
Spectroscopy to the Characterization of PEG-Stabilized Lipid Nanoparticles.
Langmuir. 2004;20(20):8839–45.
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35. Peris M. Sucrose: Properties and Determination. Encyclopedia of Food and Health.
2016. 205-210 p.
36. Yilgör I, Yilgör E, Wilkes GL. Critical parameters in designing segmented
polyurethanes and their effect on morphology and properties: A comprehensive review.
Polymer (United Kingdom). 2015. p. A1–36.
37. Hsu SH, Kao YC, Lin ZC. Enhanced biocompatibility in biostable
poly(carbonate)urethane. Macromol Biosci. 2004;4(4):464–70.
38. Ganta SR, Piesco NP, Long P, Gassner R, Motta LF, Papworth GD, et al.
Vascularization and tissue infiltration of a biodegradable polyurethane matrix. J
Biomed Mater Res A. 2003;64(2):242–8.
39. Da Violante G, Zerrouk N, Richard I, Provot G, Chaumeil JC, Arnaud P, et al.
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Chapter 3: Synthesis of biocompatible polyurethanes.
Characterization by Techniques Spectroscopy, GPC and MALDI-
TOF
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1. Introduction
The synthesis of polyurethanes has been extensively studied by several groups with the aim of
investigating the novel combination of monomers that allow obtaining polyurethanes with
greater versatility of properties (1–6).
The choice of monomers used to synthesize PUs are dependent on the final applications of the
material (7). For example, monomers that offer good durability will be selected for polymers
to be used for prostheses (8), those that increases the compatibility of PU with tissues will be
applied for pacemakers (9) and monomers that offer thermal stability will be carefully
chosen for catheters that required sterilization process (7,10). The properties of PUs are
remarkably affected by the content, type, and molecular weight of the soft segments (7).
We are interested in the synthesis of linear polyurethanes from carbohydrates suitable
functionalized for application in pharmaceutical industry. Recent work has shown that the use
of disaccharides such as sucrose increase the crosslinked of the polymer. This result is a
consequence of the number of OH in the structure of glycol. A monomer with 2 OH groups
permits obtained linear polyurethane.
Isosorbide is a monomer already used in the synthesis of biocompatible polymers(3,7,11,12).
This glycol is a chiral and relatively thermostable diol. It is technically produced from
glucose, as showed in Figure 3.1.
Figure 3. 1. Two step procedure for the synthesis of isosorbide from D-glucose adapted from
(13).
On the other hand, the PPG is a biocompatible polyether which has been extensively
investigated for application as a biomedical material that confers elastomeric properties to
polyurethanes(14–16). This monomer was then chosen as a second glycol in outlined
synthesis.
D-glucose D-glucitol D-isosorbide
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To avoid the use of monomers that, probably yield toxic compounds, the IPDI and HMDI are
envisaged as option to access to non toxic formulation since those isocyanates have been
already used in the synthesis of several biocompatible polyurethane (15,17,18).
Our work aims is to synthesize new polyurethanes from D-isosorbide by polyaddition. The
novelty of this work consists not only in the synthesis of this type of polymer but also in the
production of innovative building block to synthesize a biocompatible polyurethane.
Moreover, we established a methodology for the study of the chemical structure of this type
of PUs by FTIR, 1H NMR, 13C NMR, their molecular weight being determined by GPC/SEC
and MALDI-TOF.
2.Material and Methods
2.1 Materials
The D-isosorbide was provided by Acrös (Belgium). The HMDI was acquired from Bayer
Material Science (Germany). IPDI and DMDEE were supplied from Aldrich (Germany) and
PPG (VORANOL 1010L) from Dow Chemical (Germany). Anhydrous ethanol was acquired
by Carlo Erba (France). The DMSO-d6, CDCl3, TMS (99.9% purity), THF, Polystyrene
standards (TSK Tosoh Co), NaBF4 and DHB for MALDI-MS were purchased from Sigma-
Aldrich (Germany). The methanol (HPLC) was provided by Panreac (Spain).
2.2 Methods
2.2.1 Synthesis of quasi-prepolymers
The quasi-prepolymers were synthesized using the pre-polymerization method. As a first step,
a quasi-prepolymer was prepared by reacting of IPDI or HMDI and PPG in a 250 mL glass
flask, under a dry nitrogen atmosphere, to avoid the presence of moisture and subsequently
the formation of urea bonds during the synthesis. The reaction took place with the dropwise
addition of PPG under mechanical stirring (400 rpm), at 80 ºC and in the presence of 0.5 mL
of the catalyst (DMDEE). This reaction practically completed in 3 h. The ratios for the
syntheses are presented in Table 3.1.
.
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Table 3. 1. Molar ratios employed in the synthesis of the quasi-prepolymers.
Pre 1 Pre 2 Pre 3
IPDI 6 6 -
HMDI - - 6
PPG 1 2 1
2.2.2 Synthesis of polyurethanes
The synthesis of polyurethanes was carried out in a 250 mL reactor starting from a selected
quasi-prepolymer polyurethane. The D-isosorbide was then added to the quasi-prepolymer.
The reaction took place in 1 h. The molar ratio of the monomer is presented in Table 3.2.
Table 3. 2. Molar ratios employed in the synthesis of the PU.
PU PU19 PU20 PU21 PU22
IPDI 6 6 6 -
HMDI - - - 6
PPG 1 2 1 1
D-isosorbide 5 5 6 5
2.2.3 Determination of viscosity
The viscosity of the quasi-prepolymer polyurethanes was determined according to section
2.3.1, Chapter 2.
2.2.4 Determination of free isocyanate
The NCO content in the quasi-prepolymer polyurethanes was determined according to section
2.3.2, Chapter 2.
2.2.5 FTIR Characterization
The FTIR spectra were obtained for the quasi-prepolymer, according to section 2.2.3, in
Chapter 2. The solid samples of polyurethane were finely divided, (approximately of 1 mg)
and dispersed in a KBr matrix (200 mg). A pellet was then formed by compressing the sample
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at 784 MPa. For the sample of the liquid monomers, attenuated total reflectance (ATR-FTIR)
spectra were obtaining using the same spectrometer equipped with an ATR accessory. The
FTIR spectra were obtained at room temperature, in the range of 4000 cm-1 to 600 cm-1,
during 128 scans with 4cm-1 resolution.
2.2.6 NMR Characterization
1H-NMR and 13C-NMR spectra were obtained using a Bruker Avance III 500 MHz
spectrometer. The monomers employ in the synthesis of polyurethanes were analyzed by 1H
NMR, in chloroformed deuterated (CDCl3). However, the samples of polyurethane (PU19,
PU20, PU21, and PU22) were dissolved in DMSO-d6. All the spectra were obtained at room
temperature employing one referred internally TMS. The measurements were performed
using between 20-53 mg of samples of polyurethane. The chemical shifts are given in ppm
relative to TMS.
2.2.7 DOSY
The DOSY experiment, which is an non-invasive analytic technique, enables the
determination of diffusion coefficients, being based on the method of spin echoes with
pulsed-field gradients (PGSE) (19).
The molecular diffusion is based on the random translational movements (Brownian) of
molecules, due to their thermal energy (20). Experimentally, it appears that the probability
P(x,t) of the mass center of a molecule which diffuses from a position x to a position x+δx,
after a time t, is given by this Equation 1:
(1)
where D is the diffusion coefficient of the analyzed molecule (m2s-1). Typical values of
diffusion coefficients, at room temperature, are around 10-9-10-12 m2s-1 (respectively for little
molecules in less viscous solutions or for molecules with high molecular weight).
This technique permits to make a 2D spectrum, similar to spectrum represented in Figure 3.2,
whose represented dimensions are the chemical shift (δ) and D of the species of the sample.
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Figure 3. 2. Typical spectrum DOSY (D (m2s-1 vs δ (ppm)).
To determine the diffusion coefficients, several experiences are performed, usually varying
the strength of the gradient(21,22). In our study, the diffusion coefficients (D) were
determined at infinite dilution of the polymers. The value of D was obtained from plots of D
versus concentration. For this purpose, a series of five solutions was prepared with
concentrations 0.34×10-3 g.mL-1, 0.74×10-3 g.mL-1, 1.74×10-3 g.mL-1, 3.42×10-3 g.mL-1 and
11.4×10-3 g.mL-1 in DMSO-d6 and the diffusion coefficients D were measured using the
PGSE method in a NMR Bruker Avance III 500 MHz spectrometer with a 3 mm BBO probe
with a z-gradient shielded coil. The solutions of the polymers were poured in 3mm NMR
tubes to a total volume of 0.3mL. To guarantee reproducibility of the results this volume was
kept in all the samples. The temperature was controlled at 30°C by a BCU05 Bruker unit with
an air flow of 521 L.h-1.
The use of the exponential regression of the data allowed the determination of the diffusion
coefficient.
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The final processing and visualization of spectra was performed employing the software
Topspin, version 3.2, of the Bruker spectrometer.
2.2.8 Hydrodynamic radii (rh)
The rh for each polyurethane were determined by the Stokes-Einstein equation (Equation 2)
in which diffusion coefficient and radius are in inverse proportionality(22).
(2)
In the Equation 2,
kB: Constant of Boltzmann (1.38066 x10-23 kg.m2.k-1.s-2)
T: Temperature
: viscosity of the solution (N.s.m-2 = kg.s-1.m-1),
The Stokes-Einstein equation was originally developed for spherical colloidal particles, which
are well defined the Brownian motion. However, it is also an approach for small non-
spherical particles. The use of the equation to predict the diffusion can be improved by
replacing the radius of the molecules (r), for their effective radius rf, also called hydrodynamic
radius (rh). The value of rh can be used to compensate the factors like non-spherical molecules
and spheres of solvation.
The viscosity of DMSO-d6 at 30ºC, η = 1.951x10-3 Pa.s, was estimated from that of the non-
deuterated solvent taking into account the isotopic effects of the viscosity(23).
2.2.9 Gel Permeation Chromatographic
The analyses were made in an HPLC Waters chromatograph containing a Waters 515
isocratic pump and a Waters 2414 refractive index detector. In this apparatus, the oven was
stabilized at 35 ºC, and the elution of samples was carried out through two PolyPore columns,
protected by a PolyPore Guard column (Polymer Labs). The software Empower® performed
the acquisition and data processing.
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THF was used as eluent, at a flow rate of 1.0 mL.min-1. Before use, the solvent was filtrated
through 0.45 μm PTFE membranes Fluoropore (Millipore or Pall Corporation) and degassed
in an ultrasound bath for 45 min. The oligomer/polymer samples were also filtered across
0.20 μm PTFE filters Durapore (Millipore). Molecular weights were calibrated relative to
polystyrene standards (TSK Tosoh Co.). As a result, it should be taken into account that small
deviations could occur when the present polymer samples were analyzed.
2.2.10 MALDI-TOF
The polyurethane samples were analyzed in a MALDI-TOF mass spectrometry Voyager-
DETM PRO Workstation, equipped with a nitrogen laser operating at a wavelength of 337
nm.
A solution of polyurethane 4×10-4 mg.mL-1 in methanol was prepared (Solution 1).A second
solution of NaBF4 10 mg.mL-1 in miliQ water (Solution 2). Finally, DHB was dissolved in
Solution 2 at a concentration of 10 mg.mL-1 (Solution 3). The Solution 1 was mixed with
Solution 3 (matrix) in a ratio of 1:10 (sample:matrix), later 1 µL of the whole solution was
applied in the sample holder and allowed to dry. The resulting homogeneous solid mixture,
which ideally consists of a thin layer of microcrystals, was then introduced into the ion source
of the mass spectrometer. The time of flight mass spectrometer was in reflector and/or linear
mode. All spectra were obtained in the positive ion mode. Ionization was performed with a
337nm pulsed nitrogen UV laser. All data were processed using the Data Explorer software
package (Applied Biosystems). The spectra were averaged over 500 laser shots over the
complete sample area.
The equation of step-growth polymerization, with the name: Carothers (Equation 3) gives the
degree of polymerization. (24,25) :
(3)
Where r is the stoichiometric ratio of monomers, the excess of monomers is conventionally
the denominator so that r < 1 and, p is the extent of reaction (or conversion to polymer),
defined by Equation 4:
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(4)
Where No is the number of molecules present initially and N is the number of unreacted
molecules at the time (t). If neither monomer is in excess, then r = 1 and the equation reduces
to the equimolar. In the limit of complete conversion of the limiting reagent monomer, p → 1
The Mw (weight average molecular weight) of the polyurethanes were calculated according to
the Equation 5, and the Mn (number average molecular weight ) were calculated according to
the Equation 6, (26)
(5)
(6)
A convenient measure of the molecular weight distribution is the ratio Mw/Mn, called
polydispersity index (26).
2.2.11 In vitro cytotoxicity assay
2.2.11.1 Preparation of samples
The polyurethanes were solubilized completely in ethanol, under stirring (200 rpm), at 250
mg/mL. Later one side of the 0.5 mm diameter glass sheet was expared with this solution and
dried for 30 min.
2.2.11.2 Direct contact assay
The morphological characterization of the cell behavior in the presence of the tested materials
was performed according section 2.4.5.2, chapter 2.
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3. Results and discussion
A family of aliphatic biocompatible PU based on D-isosorbide and PPG were synthesized in
order to design several polymers to convey the active principle. The segmented PUs were
composed of various ratios of soft segments and hard segments. Polymerization of IPDI with
various ratios of D-isosorbide and PPG was carried out by poly-condensation at 80 ºC for 4h
catalyzed by DMDEE, as shown in Figure 3.3. On the other hand, correspondingly, the PU 22
was obtained from the reaction of HMDI, PPG and D-isosorbide, with the same catalyst
(Figure 3.3).
Figure 3. 3. Synthetic route of polyurethane PU19, PU20, PU21, and PU22.
3.1 Viscosity and free isocyanate of quasi-prepolymers
In the Table 3.3 the values of free isocyanate and the viscosity of the prepolymers are
presented.
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Table 3. 3. Values of viscosity and free isocyanate of quasi-prepolymers, (n=3).
Quasi-prepolymer
polyurethanes NCO/OH % NCO-
free ± SD
Viscosity
(Pa.s) ± SD
Pre – 1 6/1 12 ±0.3 10 ± 0.1
Pre – 2 6/2 9 ±0.3 21 ± 0.1
Pre – 3 6/1 11 ±0.6 10 ± 0.1
3.2 FTIR Characterization
3.2.1 Characterization of quasi-prepolymers polyurethanes
In the Figure 3.4 the FTIR of the quasi-prepolymer synthesized are presented, which
correspond to the molar ratio of Table 3.1.
600110016002100260031003600
Tra
ns
mit
tan
ce
(%
)
Wavenumber cm-1
Pre-1 Pre-2 Pre-3
Figure 3. 4. Spectra of quasi-prepolymer polyurethanes.
2252
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In all the quasi-prepolymers a band at 2252 cm-1 is observed, indicating the presence of
isocyanate groups (-NCO-) corresponding to the monomers IPDI and HMDI, which is in
agreement with the reports of the literature (2270 to 2250 cm-1 for the N=C=O stretching
vibration)(30–32). The spectra of quasi-prepolymers also showed bands at 2927 cm-1,
corresponding to -CH2- groups, and the peak of the ether group at 1527 cm-1. These bands
correspond to PPG segments, (pure PPG presents a -CH2- band between 2970-2850 cm-1 and
another one corresponding to the ether group at 1526 cm-1 (33). The band corresponding to
the carbonyl groups is observed between 1677-1708 cm-1, which is indicative of the
condensation reaction between the -OH group of PPG and the -NCO- group of HMDI and
IPDI (34–36).
3.2.2 Characterization of Polyurethanes
Polyurethanes from IDPI
The FTIR spectra of the PUs and monomers are showed in Figure 3.5.
2250
2834
3366 2973
1705-16912954 -2870 3300-3000
60012001800240030003600
Tra
nsm
itta
nce
(%
)
Wavenumber (cm-1)
IPDI Isosorbide PPG PU19 PU20 PU21
1542
1089
Figure 3. 5. FTIR spectra of Isosorbide, PPG, IPDI and PUs.
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In Figure 3.5 is observed the characteristic band of -OH- at 3366, this band correspond to the
presence of isosorbide (37). The -CH2- is observed at 2973 cm-1 corresponding to the position
mention for other authors then identified the band of methylene (37). The band OH disappears
in the spectra of PUs. The -CH2- identified in isosorbide and PPG, are detected in the spectra
of PUs between 2954-2870cm-1. The hard segment of polyurethanes present bands
characteristics, the stretching vibrations of -NH- and C=O group. The -NH- bands are
detected around 3300-3000 cm-1 and the carbonyl bands can be observed between 1705-
1691cm-1(34,38). These structures can interact and form intermolecular hydrogen bonding.
The disappearance of –N=C=O stretching vibration around 2250cm-1 in the spectra of
polyurethanes synthesized (Figure 3.4) suggested that there was no unreacted isocyanate
group (18) and meaning that all the isocyanate groups had reacted with the OH group (39).
On the other hand, NH bending bands of the PUs synthesized in our work were identified at
1542 cm-1(Figure 3.5) (31). The position of this bands was observed in the reaction between
polycaprolactone and 2-isocyanate ethyl methacrylate (40). The NH bending from urethane
group was detected too by Daniel da Silva, et al, at the same position (41). Furthermore, the
C-O-C stretching was observed at 1089 cm-1 (38).
In Figure 3.6, the intensity of some peaks in the PUs synthesized is observed.
Figure 3. 6. FTIR of PU, C=O, C–H and C–O– C stretching bands of PUs.
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The FTIR spectra of PU19, PU20 and PU21 showed differences between the intensity of
group amine –NH-, ethylene C–H stretching and ether combination absorption, band around
3344 cm-1, 2967cm-1, 2875cm-1, represented in Figure 3.5 and bands at 1718cm-1, 1542 cm-1
and 1108cm-1 presented in Figure 3.6, indicating that the molar ratio of isosorbide and PPG
influenced the spectrum intensities, that corresponding with report in the literature(7).
Polyurethanes from HMDI
In Figure 3.7 presents the FTIR of the PU22.
Figure 3. 7. FTIR spectrum of PU22.
1704
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In Figure 3.7, the peaks of CH2 stretching (from PPG) are observed between 2927 and 2854
cm-1, the band of carbonyl of urethane is detected at 1704 cm-1. The bands typically of C-N
stretching, combined with N-H in plane bending are observed at 1523 cm-1 and 1446 cm-1,
these signs demonstrating the occurrence of the reaction between the hydroxyl and the
isocyanate group (42). The C-O-C characteristic band was identified at 1079 cm-1.
3.3 1H NMR Characterization
3.3.1 Characterization of some monomer by 1H NMR spectroscopy
3.3.1. 1 Characterization of IPDI
The structure observed in Figure 3.8, show different chemical environment for all the protons
of IPDI.
Figure 3. 8. The structure of IPDI.
The spectrum of this aliphatic diisocyanate (Figure 3.8), are observed in the Figure 3.9.
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Figure 3. 9. 1H NMR spectrum of the IPDI (500 MHz, in CDCl3 at room temperature).
In the Table 3.4 was represented, the chemical shifts obtained for the IPDI as well as those
found in the literature.
Table 3. 4. Assignments of 1H NMR and chemical shifts of IPDI
Assignments Chemical shifts
CDCL3 (ppm)
Reference of chemical shifts*
CDCL3 (ppm)
1H 3.45-3.70 3.2-3.8
2H - 4H 1.45 1.4-1.9
6H 1.65-1.95 1.24-1.82
7H-8H-9H 0.85-1.25 0.9-1.3
10H 3.02 3.1-3.4
*(16,43,44)
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Usually, the IPDI consists in a mixture of cis and trans isomer, Z to E isomers in a 3:1 ratio.
The two isocyanate groups presented in the asymmetric molecule are chemically different;
one is bonded directly to a primary carbon (NCO-C10) and the other is bonded to the cyclo-
aliphatic ring to a secondary carbon (NCO-C1) (16). According to the spectrum of this
isocyanate, the assign a chemical shift of each proton is complicated.
3.3.1. 2 Characterization of PPG by 1H NMR
The structure developed for PPG (Figure 3.10) shows three chemically different protons.
Figure 3. 10. The structure of PPG.
However, when considering the chemical environment can be observed very close to each of
these types of hydrogens chemical shifts (Figure 3.10).
In Figure 3.11 is presented the 1H-NMR spectrum of PPG.
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Figure 3. 11.1H NMR spectrum of the PPG (500 MHz, in CDCl3 at room temperature).
The chemical shifts of the PPG observed are described in Table 3.5, as well as those found in
the literature.
Table 3. 5. Assignments of 1H-NMR and chemical shifts of PPG.
Assignments Chemical shifts
CDCl3 (ppm)
Reference of chemical
shifts*
CDCl3 (ppm)
Ha 1.2 1.12-1.37
Hb - H b´ 3.2-3.5 3.4-4.0
OH 3.9 3.1-3.9
* (16,45,46)
3.3.1. 3 Characterization of Isosorbide by 1H-NMR
In Figure 3.12 the structure of isosorbide and the identification of the different protons found
in the molecule is presented.
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Figure 3. 12. The structure of Isosorbide.
In the Figure 3.13 was observed the 1H-NMR spectrum of the Isosorbide
Figure 3. 13. 1H NMR spectrum of the Isosorbide (500 MHz, in CDCl3 at room temperature).
The hydroxyl of isosorbide appears at 2.2 ppm (7). Table 3.6 are presents the chemical shifts
corresponding to the structure of Figure 3.12. The peaks identified are in accordance with
isosorbide signals observed in other works for the characterization by 1H-NMR (37,47).
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Table 3. 6. Assignments of 1H NMR and chemical shifts of Isosorbide.
Assignments Chemical shifts
CDCL3 (ppm)
Reference of chemical
shifts*
CDCL3 (ppm)
1H,6H 3.8-4 3.9-4
3H,4H 3.5 3.7-4.4
2H 4.25 4.7
5H 4.6 5.0
OH 2.1 2.2
* (7,37,48,47)
3.3.2 Analysis of PUs synthesized of by NMR spectroscopy
The analysis by 1H-NMR and 13C-NMR of the PUs presented in this study disclosed four
structures.
The structure that represents the fraction of IPDI- PPG-IPDI is presented in Figure 3.14A and
the fraction of the structure corresponding to IPDI-(D-isosorbide)-IPDI is represented in
Figure 3.14B.
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Figure 3. 14. Segment of polyurethane with monomers IPDI and PPG (A). Segment of a chain
of polyurethane with monomers IPDI and D-isosorbide (B).
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The structure resulting from HMDI-PPG-HMDI is showed in Figure 3.15A and the fraction
HMDI-Isosorbide-HMDI in Figure 3.15B.
The 1H-NMR were performed in DMSO-d6 since it presented a better resolution regarding the
–NH- proton. The identification of NH indicated reaction between diols and the isocyanate
group (16).
Figure 3. 15. Segment of a chain of polyurethane with monomers HMDI and PPG (A).
Segment of a chain of polyurethane with monomers HMDI and D-isosorbide (B).
3.3.2.1 Analysis of polyurethanes by 1H-NMR spectroscopy
The 1H NMR spectra of polyurethanes PU19, PU 20, PU21 are depicted in Figure 3.16.
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Figure 3. 16. 1H NMR spectra of PUs (500 MHz, in DMSO-d6 at room temperature).
The principal signals of polyurethanes identified by 1H-NMR are summarized in Table 3.7.
The 1H-NMR spectra of the synthesized PUs synthesized are confirmed the presence of the
urethane group by the identifications of resonances of -HN-, near to 7.0 ppm. These results
are agreement with those reported by Bessel et al, where the characteristic NH- of the
urethane group,was identified at 7.1 ppm as a result of the synthesis of polyhydroxy-urethanes
by an isosorbide dicyclocarbonates from isosorbide with four commercial diamines (3). Other
authors mention that the proton from the group -NHCOO- , obtained for the reaction between
IPDI and polyester can also be observed at 6.8, 6.82 and 7.2 ppm(43). In the literature the
resonance of -NH- is described at 8.07 ppm in polyurethanes synthesized from isosorbide,
HDI, and PTMG (7).
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Table 3. 7. Assignments of 1H-NMR and chemical shifts of PU 19, PU 20 and PU21.
Assignment
Chemical
Shifts
PU19 (ppm)
Chemical
Shifts
PU20 (ppm)
Chemical
Shifts
PU21 (ppm)
Reference of
Chemical
shifts (ppm) *
1 3.80 3.90 3.75 3.20-3.80
2,4,6 1.40-1.60 1.25-1.75 1.30-1.60 1.24-1.9
7,8,9 0.70-1.10 0.60-1.20 0.80-1.20 0.90-1.30
10 2.60 2.60 2.70 3.10-3.40
11,12 7.1 7.2 7.1 7.1-8.02
13 7.30 7.00-7.25 7.00-7.50 7.00-8.07
1i 3.50 3.50 3.50 3.90-4.00
2i 4.75 4.50 4.00 4.00-4.70
3i 4.30-4.40 4.25-4.30 4.50 3.70-4.40
4i 4.40 4.60 5.00 3.70-4.40
5i 4.75 4.90 5.00 5.00
6i 4.00 3.80 4.10 3.90-4.00
a 1.20 1.00 1.20 1.12-1.37
b 3.00 3.20 3.50 3.40-4.00
b´ 3.50 3.50 3.50 3.40-4.00
c 4.90 4.80 5.00 4.70-5.00
*(7,16,43)
The spectrum of PU 22 is presented in Figure 3.17.
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Figure 3. 17. 1H NMR spectrum of PU22 in DMSO-d6. Bruker 500 MHz spectrometer.
NMR spectroscopy provided more conclusive evidence of the structure of PU 22. The
corresponding chemical shifts for 1H NMR assignment are listed in Table 3.8. The peak at 6.9
and 7.2 ppm corresponds to urethane group. The signs between 4.0-5.2 ppm are evidence of
the presence of isosorbide,in the structure of the polyurethane. The intense resonance at 1.2
ppm and 3.5 ppm shows to the presence of -CH2 from PPG, being the peaks of HMDI
identified in the range 0.75-0.9 ppm.
In the work of M Rahman et al, polyurethanes were prepared using siloxane polyol
poly(dimethyl siloxane), poly(propylene oxide glycol), poly (tetramethylene adipate glycol)
and HMDI. In the 1H-NMR spectra of these polyurethanes were identified the protons of the
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cycle of HMDI between 0.3-0.65ppm. On the other hand the -NH- of the urethane was
identified at 7.18 ppm(35,49).
In synthesis of polyether poly(urethane urea), composed of poly(ethylene glycol) as the soft
segment and 4,4′-methylenebis(cyclohexyl isocyanate) extended with ethylenediamine as the
hard segment, the urethane group was identified in 1H-NMR spectra at 7.09 and the proton
corresponds to HMDI between 0.87-1.75 ppm(50).
Table 3. 8. Assignments of 1H-NMR and chemical shifts of PU 22.
Assignment Chemical Shifts
PU22 (ppm)
Reference of Chemical
shifts (ppm) *
1a, 2a,3a,4a,5a 0.75-0.80 0.3-0.65
12a 6.90-7.20 7.1-7.2
1i 4.10 3.90-4.00
2i 4.40 4.00-4.70
3i 3.60-3.90 3.70-4.40
4i 4.75 3.70-4.40
5i 5.00 5.00
6i 3.90 3.90-4.00
a 1.20 1.12-1.37
b 3.50 3.40-4.00
b´ 3.60 3.40-4.00
c 4.80 4.70-5.00
*(7,16,35,49,50)
3.3.2.2 Analysis of polyurethanes by 13C-NMR spectroscopy
13C NMR is a powerful tool to identify monomer groups, photopolymers and copolymers
molecules of a very well defined composition.
The study of the 13C NMR spectra of the polyurethanes synthesized depicted in Figure 3.18
were relatively complex because the IPDI and the isosorbide employed in this work were used
as mixture of isomers (cis/trans). Nevertheless, the experimental chemical shifts assigned are
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according to the reference in the literature. The same phenomenon occurs in the interpretation
of PU22, synthesized from HMDI (mixture of cis–trans isomers), the spectrum of this
polymer is showed in Figure 3.19.
The structure presented in Figure 3.14 and Figure 3.15 were useful in the interpretation the
13C NMR spectra.
Figure 3. 18. 13C NMR spectra of the polyurethanes (500 MHz, in DMSO-d6 at room
temperature).
In Table 3.9 summarizes the principal chemical shifts determined for the polyurethanes (44).
The PU19, PU20 , and PU21 displayed resonances of carbonyl group between 153-157ppm.
(16).
On the other hand, the peaks of a primary isocyanate at 121.7 ppm and the secondary -NCO-
at 122.6 ppm, disappear in the 13C NMR spectra (44). These results indicate that the PUs are
free of residual IPDI.
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Table 3. 9. Assignments of 13C NMR chemical shifts for PU19, PU20 and PU21.
Assignment Chemical Shifts
PU19 (ppm)
Chemical Shifts
PU20 (ppm)
Chemical Shifts
PU21 (ppm)
Reference of
Chemical shifts
(ppm) *
Ca 16.5 16.7 16.5 17.12-18.24
C9 23.00 22.5 22.5 23.33-29.95
C7 27.9 26.8 26.7 27.19-27.71
C3 31.9 32.0 32.3 32.11-32.24
C8 34.5 34.3 34 34.92-34.97
C5 36.0 36 36 36.87-36.92
C6 - 41.5 41.5 43.63-44.13
C1 44 44 44 48.97-49.25
C4 45 45 45 46.40-46.64
C2 46 45.8 46 48.20-48.44
C10 54 54 54 51.18-57.24
Cb 68 68.7 68 72-78
C6i 70 70 70.5 70-75
C1i 71 71 71.5 70-71
Cc 72.5 72 72.5 72-78
C5i 73 73 75.5 70-80
Cb 74 74 75 72-78
C2i 75 75 75.5 70-75
C4i 81 80.5 81 80-81
C3i 85.5 85.5 86 79-81
C=O 154.3-157.5 154.3-157.5 153.3-157.5 153-157
*(16,43,44,51)
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13C NMR PU22
In Figure 3.19 is presented the 13C NMR spectrum of PU22. The urethane carbonyl resonance
are identified between 154 ppm and 155.5 ppm. This result is similar to the findings observed
in the characterization of the polyether urethane urea, prepared by reaction between HMDI
and polyethylene glycol and ethylenediamine, where the carbonyls were detected at 155 ppm
and 157 ppm (50).
Figure 3. 19. 13C NMR spectrum of the PU 22 (500 MHz, in DMSO-d6 at room temperature).
In the spectrum displayed in Figure 3.19 the resonance at 124ppm, which is characteristic of
isocyanate group, carbon atom is absent. These means that all the diisocyanate reacted (35)
which was also confirmed by the FTIR analysis (Figure 3.7).
Two strong and sharp peaks centered at 27.2 and 71.1ppm were observed in the spectrum
corresponding to the PPG structure. The signal at 51 ppm, confirmed the HMDI backbone
(35). Table 3.10 summarizes all the chemical shift from 13C NMR spectrum of the PU 22.
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Table 3. 10. Assignments of 13C NMR and chemical shifts for PU22.
Assignment Chemical Shifts
PU22 (ppm)
Reference of Chemical
shifts (ppm) *
Ca 28.00 27.00
C2a 33.00 33.00
C3a 30.00 31.00
C4a 38.00 36.0
C1a 45.00 44.00
C5a 36.0 54.00
Cb 72 71.1-72
C6i 70 70-75
C1i 71 70-71
Cc 72.5 72-78
C5i 73 70-80
Cb 74 72-78
C2i 75 70-75
C4i 81 80-81
C3i 85.5 79-81
C6a 154.0-155.5 155-157
*(16,35,43,50)
3.5 DOSY of Polyurethanes
The diffusion coefficient (D) represents the movement of a solute in a solvent. The D
basically dependent on four factors: the size and shape of the solute, the temperature, and
viscosity of the solvent(52). In the Figure 3.20 are presented the D corresponding to PUs.
The values of D measurement at different concentration are represented in Figure 3.21.
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Figure 3. 20. DOSY spectra of polyurethanes (1H 500 MHz, in DMSO-d6 at room
temperature,0.348 mg/mL).
From different concentration of polyurethanes were determined the value of D for each
polyurethane. The different measurement is observed in Figure 3.21.
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Figure 3. 21. Diffusion coefficient vs concentration for PU19‒PU22 in DMSO-d6, measured
at T = 30 oC.
In the Table 3.11, the values obtainedfor the D of the corresponding PUs are presented.
Table 3. 11. Values of D for PU19- PU22, measured at T=30oC, DMSO-d6.
The values obtained for D indicate that PU19 and PU21 presented high mobility in DMSO at
a temperature of the study. However according to expected the PU 20 showed slowly
diffusion in the solvent at the condition of the test. PU 22 also presented slow mobility,
according to the low diffusion coefficient. This result should be analyzed together with the
results of the molecular weight measurement. However, because the temperature and
viscosity of the solvent are the same in the measurements, D values depend only of: the size
and shape of the polyuretane, probably the PU 20 and PU22, present higher size and shape
that the other polyuretanes, in this condition of the study.
Polyurethane D*10-11 (m2/s-1)
PU19 7.04
PU20 4.49
PU21 6.74
PU22 4.52
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3.6 Hydrodynamic radii
The values of rh for the polyurethanes are presented in Table 3.12.
Table 3. 12. Values of rh for PU19-PU22, measured at T=30 oC, DMSO-d6
The rh according to Stokes-Einstein equation are inversely proportional to the value of D. The
rh of PU20 and PU22 are higher than the PU19 and PU21, probably they present higher size
and shape.
3.7 Result of the analyzis of polyurethanes by GPC
Table 3. 13. Average Molecular weight by GPC.
The higher number average molecular weight was obtained for the PU22, because the
polyurethane presented D = 4.52*10-11 m2/s-1. The lower number average molecular weight
was to PU21 because present higher diffusion coefficient and lower rh.
The polyurethane synthesized presented similar polydispersity index and the values are closed
to 1.
Polyurethane rh 10-9(m) Δrh 10-9(m)
PU19 1.62 0.04
PU20 2.65 0.15
PU21 1.69 0.04
PU22 2.52 0.19
Polyurethane Mw
(Dalton)
Mn
(Dalton)
Dispersity
(Ð=Mw/Mn)
PU19 15600 10300 1.50
PU20 19200 12800 1.48
PU21 9200 7200 1.27
PU22 23500 15140 1.55
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3.8 MALDI-TOF
The objectives of the use of the MALDI-TOF technique are related to the determination of the
molecular weight of the polyurethane without fragmentation.
The MALDI-TOF mass spectra of polyurethanes were obtained using DHB matrix and
sodium iodide salt as the cationization reagent, showed in Figure 3.22, Annexes 4-7 and Table
3.14. The MALDI-TOF spectra contains one mass distributions in the range 400-4200 Da
with mass intervals corresponding to the molar mass of the repeating unit (368 Mw and 408
Mw).
The PU 19 present one distribution, at m/z = 368n + 22.99(Na+) that corresponds to the D-
isosorbide and IPDI groups. This distribution allows calculated a Mw of 948.57 Dalton.
The PU 20 presents one distribution at m/z = 58N corresponding to fragment of PPG and
another at m/z = 368n + 22.99(Na+) corresponding to IPDI+ D-isosorbide. This last allows
the calculation Mw of 756.6 Dalton.
PU 21 present one distribution of chain at m/z = 368n + 22.99(Na+), that allows calculation
Mw of 975.94 Dalton.
Finally, the PU 22 present one distribution at m/z =408n + 22.99(Na+) corresponding to
fragment of PPG, HMDI + D-isosorbide, that allow calculated a Mw of 921.1 Dalton.
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Figure 3. 22. MALDI-TOF mass spectra of polyurethane after deisotoping procedure.
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Table 3. 14. Higher mass identified by MALDI -TOF and mass calculated according to molar
ratios.
Maldi-TOF
MS
Calculated
Mass
PU19 2379 3013.67
PU20 3379 4036.80
PU21 2378 3195.14
PU22 3691 3280.30
The values showed in Table 3.14 indicated that the polymer with higher molecular weight,
correspond to PU22, this agrees with the molecular weight measurement by GPC.
Nevertheless, the values of Mw for each polymer obtained by MALDI-TOF are different
compared with the determination done by GPC. The difference is justified by the
hydrodynamic volume of the polymers. The measurement of Mw obtained by GPC was
performed using polystyrene as a standard reference. The hydrodynamic volume of
polystyrene is different from the polyurethane; however, this polymer is the available
reference in GPC for polyurethane analysis.
The MALDI-TOF and GPC demonstrated, the relation between the molecular weight
expected according to the molar ratios employed in the synthesis of the polymer.
The GPC didn’t present any accuracy in the values of Mw when compared with molecular
weight determined according to molar ratios of PU19, PU21, and PU22. However, the
MALDI- TOF revealed some correspondence between the molecular weight determined and
the molecular weight calculated according the molar ratios used in the synthesis.
3.9 Cytotoxicity assay
The direct contact tests were carried out for PU19, PU20, PU21, PU22 and control slide glass.
As shown in Figure 3.23, the HaCaT shows proliferation in contact with PU19.
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Figure 3. 23. HaCaT cells after 72h of proliferation a) glass slide (control) , b) PU19.
The image obtained in Figure 3.23 show a monolayer of HaCaT in contact with the PU19
after 72h and demonstrated the compatibility of the polymer with keratinocyte cells.
From Figure 3.24 it can be observed that polymers PU19, PU20 and PU 21 presents a cell
viability (measured by the MTT reduction) that did not differ significantly from the control
(glass slide) whereas the PU22 polymer the cell viability is 80%±5% (significantly different
from control for p<0.05)
* Not significantly difference (p˂0.05), ** significantly different (p˂0,05).
Figure 3. 24. HaCaT cell viability by MTT after proliferation under different polymers
material. Control (glass slide), (mean ± SD, n = 6).
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4. Conclusions
The structure of polyurethanes was confirmed by FTIR, 1H and 13C NMR spectroscopy in all
the samples. This observation confirms the higher reactivity of the secondary NCO group of
IPDI compared to the primary group.
The NMR spectroscopy showed the properties of the polyurethane, behind the elucidation of
D and the hydrodynamic radii from DOSY. The polyurethanes with highest D, present lowest
hydrodynamic radius, in the conditions of the study.
The GPC, allowed the determination of the distribution of molecular weight according to a
standard reference. The MALDI-TOF confirms that the molecular weights determined is
according to the molar ration employed in the polyurethanes synthesized.
.
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Chapter 4: New polyurethane nail lacquers for the delivery
terbinafine – formulation and antifungal activity evaluation
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This chapter was adapted from the published paper in press:
Barbara S. Gregorí Valdes, Ana Paula Serro, Paulo M. Gordo, Alexandra Silva, Lídia
Gonçalves, Ana Salgado, Joana Marto, Diogo Baltazar, Rui Galhano dos Santos, João
Moura Bordado, Helena Margarida Ribeiro. New polyurethane nail lacquers for the delivery
terbinafine – formulation and antifungal activity evaluation. Journal of Pharmaceutical
Sciences.2017 Jun; 106(6): 1570-1577.
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1.Introduction
Tinea unguium, a fungal nondermatophytic nail infection is commonly referred as
onychomycosis. This disease is often underestimated, being considered as “only” a simple
cosmetic problem. As such, the solutions for its treatment and the investment made in
developing new ones are limited. However, this disease causes, in patients suffering from
such infection, pernicious effects on its interaction with society as well as emotional and
psychological damage (1).
One of the solutions to treat such condition is the use of nail lacquer formulations, which
emerged in the pharmaceutical market in the late 1990’s (2). Generally, they comprise the
drug dispersed in a polymer solution, which, after solvent evaporation, leaves a film on the
nail plate. Upon solvent evaporation, a polymeric water resistant film is formed that acts as a
drug reservoir, allowing the drug to permeate through the nail plate continuously (3). There
are many drugs active against Tricophyton rubrum, the dermatophytic fungus responsible for
onychomycosis (4,5). One of those drugs is terbinafine, an active antifungal drug with a
minimum inhibitory concentration (MIC) against Tricophyton rubrum of 0.004-0.06 µg/mL
(6). Nail lacquers are being considered as a solution to deliver relevant drugs to infected
nails, although there is no formulation containing terbinafine yet. The cause of this oversight
is related to difficulties in formulating vehicles using currently available polymer excipients.
The polymeric formulation must be carefully chosen since it may influence the therapeutic
nail lacquer efficacy. Chitosan-based water soluble polymers allow an excellent drug
permeation and adhesion to the nail plate, having a soft, flexible and matte finish, which may
improve patient compliance (7,8). However, this excipient leads to a faster drug release when
in contact with water, which occurs during the patient daily hygiene routines. Hydrophobic
polymers, possessing a poor solubility in water, produce more durable and harder films with a
glossier finish (9). Therefore, the development of new polymeric materials that adhere to the
nail plate and allow a continuous transungual drug delivery is still a challenge. Polyurethane
(PU) has been applied in the medical and pharmaceutical industries since 1970 in prosthesis
(3), microspheres for bone regeneration (11) and patches (12). However, the use of PU as a
vehicle for transungual drug delivery has never been explored. Aliphatic PUs are
biocompatible and present an excellent nail plate adhesion, depending on the monomers used
for their synthesis. Bachmann et al. have proven that the presence of carbohydrate monomers
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like fructose, glucose, and isosorbide as chain extenders of PUs, increases the
biodegradability of their polymers (13). Furthermore, a PU based on PEG 400 has been
synthesized by the reaction of PEG hydroxyl groups with isophorone diisocyanate, presenting
good bioadhesive properties (14). Another study disclosed a biomedical PU obtained through
the reaction of hexamethylene diisocyanate, poly(Ɛ-caprolactone) and isosorbide. Such
synthesis leads to a biodegradable and biocompatible elastomeric PU with an enhanced
affinity for cells and tissues (15).
The manuscript presented herein describes a new nail lacquer employing novel PUs
formulations. The biocompatibility, as well as the wettability and the free volume of the holes
found in the film formulations, were evaluated. Additionally, the nail lacquers’ morphology,
bioadhesion and viscosity were assessed and in vitro release tests were performed. Finally, the
in vitro antifungal activity was screened for the formulation which presented the best
properties.
2.Materials and methods
2.1 Materials
D-isosorbide was provided by Acros (Portugal). IPDI, Desmodur® I, was purchased from
Bayer Material Science (Germany) and PPG, VORANOL® 1010L, from Dow Chemical
(Germany). The DMDEE was provided by Aldrich (Germany). Anhydrous ethanol, butyl
acetate and ethyl acetate were acquired from Carlo Erba (France). Terbinafine hydrochloride
(TH) manufactured by Uquifa México S.A. was kindly offered by Generis (Portugal). Tagat®
CH 60 (PEG-60 Hydrogenated Castor Oil) was purchased from Evonik (Germany). Methanol
HPLC grade was purchased from Panreac (Spain). Triethanolamine was provided by Merck
(Germany).
2.2 Methods
2.2.1 Preparation of the PU based nail lacquers
Three PU were synthesized using the quasi-pre-polymerization method. They were prepared
by reacting IPDI and PPG in the presence of the catalyst DMDEE. D-isosorbide was added
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afterwards. The mole ratios used for each synthesis were: PU 19 (6:1:5), PU 20 (6:2:5), PU 21
(6:1:6), of IPDI, PPG and D-isosorbide, respectively as previously was described in chapter 3.
Three PU-based nail lacquer formulations containing terbinafine were prepared according the
formulas described in Table 4.1. Firstly, PUs were fully solubilized in ethanol under stirring
(200 rpm). Butyl acetate, ethyl acetate and terbinafine were added afterwards, one by one,
until their complete dissolution.
Table 4. 1. PU, terbinafine based nail lacquers composition.
Formulation
A PU19 -10%
TH
Formulation B
PU 20-10%
TH
Formulation C
PU 21-10%
TH
PU 19 10.0 -- --
PU 20 -- 10.0 --
PU 21 -- -- 10.0
Terbinafine HCl 1.0 1.0 1.0
Ethyl acetate 7.8 7.8 7.8
Butyl acetate 10.0 10.0 10.0
Ethanol 71.2 71.2 71.2
2.2.2 In vitro cytotoxicity assay
The biocompatibility of the PU based nail lacquers was evaluated in vitro by direct contact
with cells, following the ISO 10993-5:2009 recommendation guidelines (16). Each nail
lacquer formulation material was added to a 0.5 mL of HaCaT cell suspension [a
spontaneously immortalized human keratinocyte cell line (CLS, Germany)], in fresh culture
medium [RPMI-1640® (Gibco, UK) medium supplemented with 10% fetal bovine serum
(FCS, Life Technologies, Inc., UK), penicillin (100 IU/mL), and streptomycin (100 μg/mL)]
(2.5 x 104 cells/mL), in sterile 24-well plates. Glass slides without any formulation were used
as control. The plates were incubated for 72 h in a humidified atmosphere of 5% CO2 at 37
°C, without refreshing the culture medium. For cell proliferation quantification, the general
cell viability endpoint MTT reduction (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide) was used (17,18). Accordingly, the previous culture medium was
removed and replaced with a fresh medium containing 0.5 mg/mL of MTT. The cells were
further incubated for 3 h. In the plates containing the reduced MTT, the medium was removed
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and the intracellular formazan crystals were solubilized and extracted with dimethylsulfoxide.
After 15 min, at room temperature, the absorbance was measured at 570 nm in a Microplate
Reader (FLUOstar Omega, BMGLabtech, Germany).
The data are expressed as the mean and the respective standard deviation (mean ± SD) of 6
experiments. The statistical evaluation of data was performed using one-way analysis of
variance (ANOVA). The Tukey–Kramer multiple comparison tests (GraphPad PRISM 5
software, USA), were used to compare the significance of the difference between the groups;
a p < 0.05 was accepted as statistically significant.
2.2.3 Determination of wettability by measurement of contact angle
The measurement of the static contact angle of water with the therapeutic nail lacquer samples
was carried out by the sessile drop method (19).
Drops (2 – 4 L) were generated with a micrometric syringe and deposited on the substrate
surface, inside a temperature-controlled chamber (25 °C), which had previously been
saturated with water to avoid the drops’ evaporation. A sequence of images, obtained with a
video camera (JVC Colour) mounted on a microscope (Wild M3Z) and connected to a frame
grabber (Data Translation DT3155), was recorded during 60 s, starting from the moment of
the drop deposition. Images were analyzed using the axisymmetric drop shape analysis-profile
(ADSA-P) program. At least 6 drops were analyzed on the air-facing surfaces of each film.
2.2.4 PALS
The PALSs of the nail lacquer formulations films were determined by detecting the prompt -
ray (1.28 MeV) from the nuclear decay that accompanies the emission of a positron from the
22Na radioisotope and the annihilation -ray (0.511 MeV). A fast-fast coincidence circuit of
the PALS setup (featuring Pilot-U scintillators and XP2020 photomultipliers), with a time
resolution of approximately 260 ps (FWHM), was used to record the positron lifetime spectra.
The positron 22Na source (ca. ~10µCi, closed between Kapton foils) was sandwiched by two
identical samples. Several spectra were collected at room temperature. The lifetime spectra
had a total number of ca. 4x106 integral counts and were evaluated using the LT (version 9)
software (20,21).
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2.2.5 SEM
The SEM Field Emission Gun (FEG) SEM JEOL JSM-7001F was used to obtain images of
the surface of each therapeutic nail lacquer formulation film. Human nails were obtained with
nail clippers from the fingers of a healthy Caucasian female of 24 years of age, after ethical
approval and informed consent. The nail plates were cut 24 h before each test. The nail plates
were stored in a glass jar with paper moistened with distilled water to avoid dehydration.
First, the formulations were applied on the nails, by using a thin rectangular plastic rod (3 x
0,5 mm). The nails were dried at room temperature. The samples were gold sputter coated (10
nm) in a chromium deposition equipment (Q150T ES, Quarum Technologies).
2.2.6 Adhesion test
The assessment of the adhesion of therapeutic nail lacquers was developed as an adaptation of
ISO 2409:2013(22). This manual measurement of film adhesion is used to measure the
resistance of paint coatings with a Kit Elcometer® 107. A quantity of 0.25 ± 0.02 g of the
therapeutic nail lacquers were weighed and spread in cow horn samples, to create a
homogeneous film. Cow horn samples were cut in cylinders 35 mm in diameter, 1.5 mm
thick. The samples were polished with a 600 mesh SiC paper under flowing water for
lubrication, and then rinsed with water. Afterwards, the samples were dried with absorbing
paper and stored at room temperature. The nail lacquer formulations were applied on the cow
horn samples and dried at room temperature for 15 min. Subsequently, the samples with nail
lacquer films were cut in a lattice pattern with a cutting blade (Kit Elcometer® 107, France).
The first cut was made at 45o angle to the direction of the grain and the second cut was
repeated at 90o angle to the first. Afterwards, the sample was brushed (Kit Elcometer® 107,
France). Then, an adhesive tape (ISO 2409 Elcometer® 107, France) with the size of the cow
horn samples, was placed over the lattice pattern. The adhesive tape was removed at an angle
of 60o with the surface, and the latter was examined and evaluated according to the
classification of the ISO 2409:2013 guideline, where 5 refers to ‘very damaged’, meaning the
lattice pattern and the sample have bad adhesion, and 1 refers to a value for good adhesion,
corresponding to the integrity of the lattice pattern. The test was repeated 5 times for each
therapeutic nail lacquer.
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2.2.7 Viscosity measurement
The determination of viscosity was performed in a cone-plate Brookfield® viscometer, model
DV-II + Pro coupled with a constant temperature bath, previously gauged with a CPE-40
spindle. The temperature of the measurement was 32 ± 0.2 oC, shear rate 375 s1 and shear
stress 0.8-1 N/cm-2. A volume of 500 µL of therapeutic nail lacquer was used for this test.
2.2.8 In vitro release of terbinafine hydrochloride from therapeutic nail lacquers
For the in vitro release studies of terbinafine release from the therapeutic nail lacquers, 200-
300 mg of each formulation (equivalent to 2-3 mg of terbinafine hydrochloride) was weighed
in glass slides, dried and suspended in 15 mL of a 0.5% Tagat® CH 60 aqueous solution.
Containers were placed in an incubator-shaker at 32 ± 0.5 °C for 24 h. Samples of 1000 µL
were collected at predefined times (1, 2, 3, 4, 8 and 24 h) and the same volume was replaced
with fresh receptor solution maintained at the same temperature.
The samples of terbinafine were analyzed by HPLC-UV in a Hitachi Elite Lachrom System
(VWR, USA) equipped with four L-2130 pumps, an autosampler L-2200, a column
Lichrospher 100 RP18 (150 mm x 4 mm, 5 µm, Merck), a UV Detector L-2400 and a signal
processing software EZ Chrom Elite Version 3.2.1. The method used an isocratic gradient
mobile phase containing 0.5% (v/v) triethanolamine, 84.5% (v/v) methanol and 15% (v/v)
water. A flow rate of 1.0 mL/min was used with a 10 µL injection volume. The auto sampler
chamber was maintained at room temperature and the eluted peaks were monitored at a
wavelength of 283 nm. The run time was 5 min. All chromatographic separations were carried
out at 25 ºC. A calibration curve was performed using 19.8 µg/mL, 39.7 µg/mL, 79.4 µg/mL,
99.2 µg/mL and 119 µg/mL of terbinafine hydrochloride in methanol. Three replicates per
formulation were analyzed.
The data obtained from the in vitro release studies was computed using a DDsolver (23),
which is an Excel-plugin module, and the obtained data were fitted to four different kinetic
models: zero order, first order, Higuchi and Korsmeyer-Peppas24. For all models, the adjusted
coefficient of determination (R2 adjusted) was estimated, fitted and used as the model ability to
describe a given dataset.
Statistical analysis was performed on the release profiles obtained using an ANOVA two
factor with replication, assuming p values ≤ 0.05 as statistically significant.
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2.2.9 In vitro antifungal activity
Candida albicans ATCC 10240, Aspergillus brasiliensis ATCC 16404 and Trichophyton
rubrum ATCC 28188 were used for the determination of in vitro antifungal inhibitory activity
of a terbinafine nail lacquer formulation. The test was performed according to the standard
disc diffusion method (DDM) in accordance with the Clinical and Laboratory Standards
Institute (CLSI) guidelines (25). The Mueller-Hinton agar (BioKar, Spain) microbiologic
medium was used.
The test took place over 2 days for Candida albicans and 7 days for Aspergillus brasiliensis.
For Trichophyton rubrum the study took place over 20 days under 22.5 ± 2.5 ºC and humidity
≥ 85%. The positive or negative activity of the formulation was assessed according to the
presence or absence of growth around the disks and is an indirect measure of the ability of the
formulation to inhibit those microorganisms. Fungal inoculum: stock inoculum fungi
suspensions were prepared from 5 –7 days old cultures grown on Mueller-Hinton agar. The
suspensions obtained were diluted with distilled water to obtain a turbidity equivalent to a
McFarlard 0.5 turbidity standard (25), containing 1.5 x 108 cfu/mL of microorganisms. 20
mL of the Mueller-Hilton agar mediums were poured into petri dishes (90 mm diameter) and
inoculated with 100 µL of the suspension containing 1.5 x 108 cfu/mL of each microorganism.
Sterile paper discs (6 mm, Sigma-Aldrich, UK), were loaded with 15 µL of Formulation A PU
19 with a concentration of 10 mg/mL. Placebo formulations were used to compare the
inhibition zones. The antifungal activity of the formulations was compared to the antifungal
activity of a commercial terbinafine cream, Lamisil® 1%. All the results were recorded by
measuring the zone of growth inhibition surrounding the discs. All determinations were made
in triplicate.
3. Results and Discussion
3.1 In vitro cytotoxicity
The direct contact assay tests were carried out for the nail lacquer formulations and control
glass slide. No significant morphologic changes were observed in the HaCaT cells in contact
with any of the tested materials for all the studied time periods (results not shown). All nail
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lacquer formulations presented values superior to 70% cell viability (Figure. 4.1), thus it can
be concluded that these materials are biocompatible according to this viability assay.
0
20
40
60
80
100
120
140
Control Formulation APU 19-10% TH
Formulation BPU 20-10% TH
Formulation CPU 21-10% TH
% C
ell
viab
ility
Samples
Figure 4. 1. HaCaT cell viability by MTT after proliferation under different nail lacquer
formulations. Control (glass slide) (mean±SD, n = 6).
The evaluation of the formulations’ toxicity over cells surrounding the application site
according to the ISO 10993-5:2009(16), is an essential test for the regulatory authorities of
the pharmaceutical industry. Nail lacquer formulations are not in contact only with the keratin
of the nail plate, but also come into contact with the skin.
The keratinocyte cell line was selected to evaluate the cytotoxicity of the nail lacquer
formulations containing terbinafine, since these cells are the predominant cell type in the
epidermis, the outermost layer of the skin, comprising 90% of the epidermic cells, thus
presenting an adequate in vitro cell model that better mimic in vivo conditions (9). After
analyzing the nail lacquer formulations by direct contact, a monolayer of HaCaT cells was
observed, hence the conclusion that the formulations are biocompatible. These results are in
agreement with other applications of PU polymers that demonstrated their biocompatibility in
HaCaT cell line for proteins delivery (34) and in L929 cell line for other applications (35).
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3.2 Wettability
The presence of water in the nail is related to the diffusion of drug through the nail (9) and for
this reason it is important to measure the wettability of nail formulations.
A water contact angle lower than 90° indicates that wetting of the surface is favorable and that
the fluid will spread over a large area on the surface. On the other hand, contact angles greater
than 90° mean that wetting of the surface is unfavorable; the fluid will minimize its contact
with the surface and form a compact liquid droplet (26).
The average values of the angles obtained were: Formulation A PU 19-10% TH, ɵ = 45 ± 6°,
Formulation B PU 20-10% TH, ɵ = 77 ± 4° and Formulation C PU 21-10% TH, ɵ = 61 ± 4°.
The nail presented a contact angle of ɵ = 94 ± 8°. The one-way ANOVA statistical test
highlighted a significant difference between the mean values of the different PUs and the nail.
The wettability test results showed water contact angles lower than those observed for the nail
surface alone, which presented a reference value of 94º. In the work of Mândru et al., the
polymers synthesized with poly (ethylene glycol), poly (butylene adipate), methylene
diphenyl-diisocyanate and 1,4-butane-diol, presented contact angle values of 73º and 87º. This
result explained the hydrophilic properties of the nail lacquer preparations, which contained
PU with polyester and derivatives of carbohydrates in their chain structures (36).
3.3 PALS determination of free volume in films
This technique is the most efficient method for studying subnanometer size distributions and
free volume fractions, which has been used to explain the free electron property, structural
relaxation, mechanic deformation, permeability and physical aging of polymers (27,28,29,30).
PALS was used to assess the free volume of the formulations. This non-destructive technique
probes local free volumes between molecular chains in polymeric structures. In this
technique, the anti-electron, i.e. the positron, is employed as a probe and monitors the lifetime
of the positron and Positronium, Ps (a bound atom which consists of an electron and the
positron), in the polymeric materials under study (31). Due to the positron’s positive charge,
itself and the Ps are repelled by the polymers core electrons and trapped in open spaces, i.e.
the free volume. The trapped Ps could appear either as a para-Positronium (p-Ps, spin singlet
state) or as an ortho-Positronium (o-Ps, triplet spin state), with a relative abundance of 1:3,
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respectively. The annihilation photons come from these open spaces mainly, and the results of
the positron annihilation lifetime (PAL) measurements give evidence that the positron and the
Ps are located in these preexisting free volumes in polymers. In PAL measurements, the
observed lifetime () is the reciprocal of the integral of the positron and the electron densities
at the site where the annihilation takes place (31). A larger hole, which has a lower average
electron density, is expected to have a longer Ps lifetime. A correlation between the free
volumes in molecular systems and the observed o-Ps has been postulated (32,33). This
correlation is expressed in a semi-empirical equation (Equation 1) (33) between the o-Ps
lifetime ( ) and the mean radius of holes (R):
(1)
where and R are in the units of nanosecond and angstroms, respectively, and ΔR (= 1,66
Å) is the best fitting parameter between observed o-Ps lifetimes and known mean hole radii in
porous materials. With equation 2, the mean free volume hole size in a polymeric material can
be determined, by measuring the o-Ps lifetime. As the holes are assumed to have a spherical
shape, this lifetime is related to a mean radius R. With this assumption, the correspondent
free-volume cavity was calculated with the equation V = (4/3)R3. The intensity of the o-Ps
lifetime component, , is often treated as a measure of the density of the holes31. The free
volume fraction (Fv), Equation 3, is directly related to and through the following
expression (31):
(3)
Where C is an empirical scaling constant that reflects the probability of o-Ps formation.
In the systematic PAL analysis it was observed that all spectra were well fitted with the
components showed in Table 4.2; two short-lifetime components of around 200 ps and 420-
470 ps, which could be assigned to the annihilation of free and trapped positrons,
respectively, and the longest lifetime component (>2100 ps) was due, of course, to the pick-
off annihilation of o-Ps in the free volume holes. The values of the lifetime components and
the intensities of the positron and o-Ps annihilation are presented in Table 4.2.
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Table 4. 2. PALS parameters (lifetime and intensity components), hole radius (R) and free
volume cavity associated with the o-Ps lifetime for PU, terbinafine based nail lacquers.
Samples 1
(ps)
I1
(%) 2
(ps)
I2
(%) o-Ps
(ps)
Io-Ps
(%)
Volume
(Å3)
Formulation A
PU19-10% TH 196.8±7.6 27.2±2.4 436.2±9.8 46.0 ±2.4 2190.0±1.0 26.8±0.1 114.2±1.1
Formulation B
PU20-10%TH 212.8±6.6 32.2±1.8 470.5±9.4 39.5±1.9 2490.0±7.0 28.3±0.3 145.1±1.3
Formulation C
PU21-10%TH 192.0±3.1 26.5±1.0 420.7±4.2 46.5±1.0 2153.0±5.0 26.9±0.2 110.8±1.1
The free volume concept is extensively adopted in polymer science to explain the change of
microscopic structure and to relate it to macroscopic properties. In the last decades, PALS
measurements have revealed that a strong correlation exists between the free volume of
polymers and the possibility of charging them for sustained release or for selection
permeability (38).
The free volume of the tested formulations was determined using PALS: the lifetime of o-Ps
was used to define the size of free volume in the molecular system (holes) of the films and the
intensity of the o-Ps lifetime , to quantify the density of holes. The PALS results are
summarized in Table 4.2. The two short-lifetime components of around 200 ps and 420-470
ps are assigned to the annihilation of free and trapped positrons, respectively. The longest
lifetime component (>2100 ps) was due to the pick-off annihilation of o-Ps in the free volume
holes. The o-Ps lifetime component and its intensity are the only parameters significantly
sensitive to the change in the free volume and all attention must be given to the variation of
these parameters. The values of the lifetime component and the intensity of the o-Ps for these
formulations revealed that Formulation A PU19-10%TH and Formulation C PU21-10%TH
had a similar free volume; indeed, both the radius of the holes (R) and the density of holes
( ) were similar. On the other hand, Formulation B PU20-10%TH showed,
simultaneously, higher values for and , which were considered to be associated
with an increase of the free volume fraction of this polymer, compared to Formulation A PU
19-10% TH and Formulation C PU 21-10%TH. The PU19 and PU21 had identical molar
ratios of PPG in the process of synthesis, while the PU20 had a higher molar ratio of PPG.
The observed amorphous characteristic of PU20 was corroborated with the increase in the free
volume fraction of Formulation B PU 20. The mean radius of the cavities of this formulation
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was higher than Formulation A PU 19-10% TH and Formulation C PU 21-10%TH.
Additionally, the value of was also higher, revealing an increase of the number of
cavities in these films.
3.4 SEM
Images of a nail without varnish (A) and three nails containing the three developed
formulations were obtained by scanning electron microscopy (SEM) (Figure. 4.2). It was
observed that the nail lacquers leave a homogenous film covering the entire nail, making the
surface of the nails uniform.
Figure 4. 2. Scan Electron Micrograph with 1000x magnification. A) dorsal surface of nail
plate B) film of Formulation A PU 19-10%, C) film of Formulation B PU20-10%, D) film of
Formulation C PU21-10%.
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The nail lacquer films’ elucidated morphology, by SEM, revealed them to be homogeneous
and smooth. The application of the nail lacquers improved the homogeneity of the nail
surface, making it smooth and uniform. The nail lacquer film homogeneity, porosity and
distribution were similar for the three nail lacquers. Nevertheless, Formulation A PU 19-
10%TH and Formulation C PU 21-10%TH presented a thinner film with almost no pores,
while Formulation B PU 20-10% TH showed visible pores and cracks, on the surface (Figure.
4.2 C). This was related to the amorphous characteristics of PU20, presented in formulation
B. The presence of terbinafine hydrochloride had no influence in the homogeneity of the films
obtained after the formulation had been applied on the nails.
3.5 In vitro adhesion test
All the PU terbinafine nail lacquers adhered to the keratin of cow horn. The values of flaking
in the lattice pattern for Formulation A PU 19-10% was 1.8 ± 0.5 and for Formulation C PU
21-10% TH the value was 0.7 ± 0.5, which means that both formulations presented good
adhesion to the keratin of cow horn. On the surface of the cross-cut area some detachment of
small flakes of the coating could be observed where the cuts were intersected. In the case of
Formulation B PU 20-10%TH, the value was greater than 5.0 ± 0.5, corresponding to a
appearance of surface of cross-cut greater than 65 %.
The nail lacquer film must adhere to the nail plate. The adhesion test procedure examines the
cut area and the results obtained are compared to a six-step classification. The first three steps
classification are satisfactory for general purposes and are used when a pass/fail assessment is
required.
Regarding Formulation A PU 19-10%TH and Formulation C PU 21-10%TH, the obtained
adhesion values showed some detachment of small flakes of the coating at the intersections of
the cuts, on the surface of the cross-cut area. These results are promising when compared to
previous studies that assessed the adhesion of a UV-curable gel formulation with
methacrylates; in this study, the coating flaked along the edges of the cuts partly in large
ribbons and some squares detached partly or wholly, corresponding to higher mean cross-cut
score values (3 and 4) (38).
In the case of Formulation B PU 20-10%TH, the value corresponded to a classification of 5,
which means the coating flaked along the edges and at the intersections of the cuts. The PU
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terbinafine nail lacquer that presented the higher flaking in the lattice pattern was Formulation
B PU 20-10%. This means it is not effective in adhering to the keratin of cow horn. On the
other hand, the formulation with better adhesion to cow horn was the Formulation C PU 21-
10%TH, with a degree of flaking in the lattice pattern of 0.7. The increased values of
adhesion are thought to be related to the proportional increase of isosorbide in the PU chain,
due to the formation of hydrogen bonds between the NH groups of the cow horn’s keratin and
the oxygen of the isosorbide sub-structure in the polymer.
3.6 Viscosity values
The viscosity values for the therapeutic PU terbinafine nail lacquers at 32 oC were: 2.62 ±
0.04 mPa.s for Formulation A PU 19-10%; 2.67 ± 0.98 mPa.s for Formulation B PU 20-10%;
and 3.42 ± 0.07 mPa.s for Formulation C PU 21-10%. There are no significant differences
among the formulations, probably due to the fact that viscosity was determined at the same
temperature used for the study of drug release (32 ± 0.5 °C); the aim was to mimic nail
lacquer application.
3.7 In vitro release of terbinafine from Formulations A PU 19-10%, B PU 20-10% and C
PU 21-10%
Figure 4.3 shows the release profiles of terbinafine hydrochloride from the PU based nail
lacquers for 24 h.
Figure 4. 3. Release profile of terbinafine from PU based nail lacquer for 24 h in aqueous
solution of 0.5% Tagat® CH 60 at 32ºC (mean ±SD, n = 3).
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All the formulations released the drug from the matrix. Additionally, the release data were
fitted to different mathematical models to describe the release profile. The best-fit was
obtained with the Korsmeyer-Peppas function for Formulation A PU 19-10%TH and
Formulation C PU 21-10% TH and Higuchi function for Formulation B PU 20-10%TH; as
confirmed by the highest values for R2adjusted and model selection criterion (MSC) and the
lowest values for Akaike Information Criterion (AIC), they statistically described the best
drug release mechanism (23,24).
Concerning the release results, Formulation B PU 20-10%TH released the highest amount of
drug (32.4%) in 24 h, followed by Formulation A PU 19-10%TH (17.3 %) and Formulation C
PU 21-10%TH (2.6%). Once again, the polymer composition is an important factor, because
it influences the diffusion of the drug from the nail lacquers. Formulation B PU 20-10% TH
contained PU with a high PPG content, which permitted the incorporation of the drug in an
amorphous state. The degree of solvent permeability of Formulation B PU 20-10%TH,
determined by PALS, resulted an increase of released terbinafine due to a higher water
permeability (39). Formulations A PU19-10% and C PU21-10%TH, showed a delayed release
of terbinafine in the tested time range. Therefore, the contact with the receptor solution was
lower and the holes’ radii in these formulations reduced the permeation of solvents, leading to
a lower diffusion of the drug through the nail plate. All release profiles suggested a diffusion-
controlled release over the tested time period. According to the kinetic models, the diffusion-
controlled release behavior was due to the dispersion of terbinafine in a homogeneous and
uniform matrix, which acted as the diffusional medium, indicating a uniform drug distribution
over the PU matrix (40,41).
However, as formulation C PU 21-10%TH has more hydrophilic groups, leading to a more
reticulated network (a gel) which does not allow the drug to be released.
3.8 Antifungal activity
The antifungal activity of the drug against Trycophyton rubrum, Candida albicans and
Aspergillus brasiliensis was determined in terms of the mean diameter of zone inhibition. The
results obtained are shown in Table 4.3. Formulation A PU 19-10% was proved to be more
effective (higher inhibition halos) for the Trycophyton rubrum and Candida albicans when
compared to the commercial cream.
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Table 4. 3 Antifungal activity of different formulations with terbinafine against Trycophyton
rubrum, Candida albicans and Aspergillus brasiliensis.
Inhibition zone (mm)
Formulation A
PU19-10%TH
Formulation B
PU20-10%TH
Formulation C
PU21-10%TH
Solution of
terbinafine 1%
Commercial
Cream
Candida albicans
ATCC 10240 38.4±3.6 26.7±0.5 27.3±2.6 21.4±1.8 < 6
Aspergillus
brasiliensis
ATCC 16404
25.0±0.4 26.8±1.7 24.4±0.6 32.0±0.8 27.0±0.1
Tricophyton
rubrum
ATCC 2818
90.0±0.3 n.d. n.d. n.d. 26.6±0.1
n.d: not determined.
The Minimum Inhibitory Concentration (MIC) is the lowest drug concentration that prevents
microbial growth. A lower MIC value indicates that less drug is required for inhibiting the
growth of a microorganism. Therefore, drugs with lower MIC scores are more effective. The
reference terbinafine MIC values vary from 0.004 to 1 µg/mL against Tricophyton rubrum
(6), Candida albicans and Aspergillus brasiliensis (42). All formulations presented
antifungal activity (Table 4.3). The controls presented inhibition zone ˂ 6 mm.
The antifungal activity results demonstrated that Formulation A PU 19-10%TH showed
activity against dermatophytes and non-dermatophytes. In addition, Formulation B PU20-
10%TH, Formulation C PU21-10%TH showed lower inhibitory effects against Candida
albicans in compared to Formulation A PU 19-10%TH.
Although the activity substance concentration is the same in all preparations, the commercial
reference (Lamisil® 1% cream) is a formulation with higher viscosity values which may
influence the release of terbinafine and consequently the microbiological efficacy.
The Formulation B PU 20-10%TH showed the higher antifungal activity against Aspergillus
brasiliensis. However, Formulation A PU 19-10%TH presented higher antifungal activity
against Candida albicans, probably by the influence of -NH- of polyurethane in the pH of the
medium that potential the antifungal activity of the drug. The in vitro activity of terbinafine is
pH dependent and rises with increasing pH value (43).
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4. Conclusions
In the present work, three nail lacquer formulations employing polyurethanes for the release
of terbinafine were characterized. Relevant properties for the intended application were
studied, namely cell viability, wettability and free-volume. All the formulations were
biocompatible with keratinocytes and presented hydrophilic properties. The PALS study
indicated that the nail lacquer formulations have an unusually high free-volume. The nail
lacquers showed potential to adhere to the nail plate, and the formulation film (after
application) formed a homogeneous layer of terbinafine hydrochloride, according to images
obtained by SEM and the mechanical test.
These studies contributed to the development of new nail lacquer formulations with adequate
properties for controlled drug release, nail adhesion and biocompatibily for the treatment of
onychomycosis. The antifungal activity of the nail lacquer formulations were demostrated.
The Formulation A PU 19-10%TH allow more effective action against Trycophyton rubrum,
Candida albicans and Aspergillus brasiliensis.
Furthermore, it contributed to the fine-tuning of a novel controlled drug release system: PU
based nail lacquers, containing terbinafine hydrochloride as a model drug.
The novel system showed potential to be further developed as a topical polyurethane based
nail lacquer for the treatment of onychomycosis. It is expected that this novel system may be
used to release many other lower molecular weight, ionic drugs to keratinous surfaces.
According the results obtained new formulations must be performed studing the content of
PU19.
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Chapter 5: Formulation optimization of PU based Nail Lacquers
containing Terbinafine hydrochloride and Ciclopirox Olamine
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1. Introduction
Onychomycosis is a nail fungal infection caused by dermatophytes, non-dermatophytes and
yeast species(1).
Candida species have a high incidence in fingernail infection, present in as many incidence of
as 75% of cases, and are more prevalent than dermatophytes(2). In contrast, the incidence of
yeasts in toenail infections is much lower; approximately 2-10% cases. Infection attributed to
non-dermatophytes are estimated to be 2-65% of cases although higher rates, 15% have been
reported(3).
The untreated onychomycosis may worsen, spread to other uninfected locations (other nails or
to the surrounding skin) or infect other patient (4).
Nail keratin is impermeable structure thus restricting drug access to the organisms causing
onychomycosis(5). The development of therapeutic transungual drug delivery is an urgency
for patients that are affected by such infection.
Topical treatment would be of special interest for immunosuppressed, diabetic and elderly
patients who suffers from chronic pathologies and follow long-term drug therapies and among
whom the prevalence of onychomycosis is higher(5,6). Despite an active interest in method
aimed at improving the efficacy of such formulations and the permeation study is testified by
recent research report (7–10).
Many antifungal formulations have been developed. Some of them are related with ciclopirox
olamine and terbinafine hydrochloride.
The ciclopirox olamine (CPX) is used in nail topical formulations have been marketed
worldwide for about 25 years and it is available in different pharmaceutical preparations:
creams (Selergo®, Mycoster®, 1%), lotion (Mycoster®, 10mg/L) (11), gel (Loprox®, 0.77%)
(12) and solutions, such as Penlac®, Batrafen®, Mycoster®, Ciclopoli®, Ony-Tec® and
RejuveNail® containing 8% of active substance. They present butyl monoester of poly methyl
vinyl ether/maleic acid (5) or hydroxy propyl chitosan as film formers (13). The CPX present
a minimum inhibitory concentration value for Candida albicans and Aspergillus species
between 0.13-4 µg/mL (14,15).
On the other hand, terbinafine hydrochloride (TH) is used in systemic and topical
formulations (Lamisil® cream, 1%, Mycova® nail coat, 10%, TDT 067 liquid spray, 15
mg/mL and P-3058, hydroxypropyl chitosan based, 5%) (5,16). These nail formulations uses
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dodecyl 2-(N,N dimethylamino)-propionate hydrochloride, soybean phosphatidylcholine and
hydroxypropyl chitosan, respectively (5). TH, an allylamine derivative, represents the most
effective antimycotic drug, presenting a minimum inhibitory concentration against
dermatophytes of 0.004-0.06 µg/mL(17), non-dermatophytes of 0.063-2.5 µg/mL(18) and
yeast of 0.06-8 µg/mL(18)
All of them have proved to be effective presenting transungual permeation. However, there is
a need to improve the patient compliance with less repeat applications meaning that topical
nail’ formulations needs to be improved. Nail lacquers, formulated with polymers that act as
film former agents, could be an alternative to be used as of new drug carriers(9).
In this study, the influence of polyurethanes (polymer) concentration on the properties of nail
lacquer formulations is assessed and compared with a commercial formulation. The
incorporation of two drugs (TH and CPX) validated the use of PU as versatile polymers in
nail lacquer formulations. Direct contact cytotoxicity, wettability, SEM, adhesion, drying
time, viscosity measurement, permeation studies and antifungal activity were performed in
order to achieve these aims.
2. Material and methods
2.1 Materials
Ethanol anhydrous, butyl acetate and ethyl acetate were acquired from Carlo Erba (France).
The terbinafine hydrochloride manufactured by Uquifa México S.A. was kindly offered by
Generis. Tagat® CH 60 (PEG-60 Hydrogenated Castor Oil) was a kind gift from Evonik
(Germany). PU 19 was synthesized in laboratory of IST according to described in the chapter
3. Ciclopirox was obtained from Fagron Iberica (Spain), Ony-Tec® was kindly offered by
Laboratorio Medea, Reig Jofre (Spain).
2.2 Methods
2.2.1 Preparation of Nail lacquers
The PU 19 was the polymer selected for preparing 5 different nail lacquers formulations
containing 1% of drug (TH and CPX). Different polymer concentrations (10%, 15%, 20% and
25%) were employed. The formulations were prepared according to component described in
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Table 5.1. Firstly, polyurethanes have been fully solubilized in ethanol under stirring (200
rpm). Afterward, the solvents and the drugs (TH and CPX) were added, separately, until its
complete dissolution. Placebos, formulations with no drug, were also prepared.
A commercial formulation containing 8% (w/v) of CPX was also used as a control: Ony-
Tec®. It´s qualitative composition is ethanol, water, ethyl acetate, hydroxipropyl chitosan and
cetylstearyl alcohol.
Table 5. 1. Nail lacquers composition.
Formulation A
PU19-10%
TH
Formulation D
PU 19-15%
TH
Formulation E
PU 19-20%
TH
Formulation F
PU 19-25%
TH
Formulation G
PU19-10%
CPX
PU 19 10 15 20 25 10
Terbinafine
HCl (TH)
1.0 1.0 1.0 1.0 -
Ciclopirox
(CPX)
- - - - 1.0
Ethyl
acetate
7.8 7.2 6.8 6.3 17.8
Butyl
acetate
10 9.6 9.0 8.5 -
Ethanol 71.2 67.2 63.2 59.2 71.2
2.2.2 Direct contact cytotoxicity assay for Nail lacquer
The cytotoxicity test are described in chapter 3, section 2.2.11.
2.2.3 Determination of wettability by contact angle measurement
The contact angle measurement is described in chapter 4, section 2.2.3
2.2.4 SEM
The nail morphology study before and after nail lacquer formulations were applied are
described in chapter 4, section 2.2.6.
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2.2.5 Adhesion test
The adhesion of nail lacquers test is described in chapter 4, section 2.2.7
2.2.6 Drying time for nail lacquer therapeutics
The nail lacquer must dry. A quantity of 0.25±0.02g of the nail lacquers were weighed and
spread into a glass, to create a homogeneous film. The drying time was measurement with a
chronometer. This methodology was adapted from ISO 2409:2013(19).
2.2.7 Viscosity determination
The viscosity determination is described in chapter 4, section 2.2.7.
2.2.8 In vitro release of terbinafine from nail lacquers containing different
concentrations of PU
In vitro release studies were performed using Franz diffusion cell apparatus through a
hydrofilic membrane (Tuffryn® Membrane) (Pall corporation 79579I), with a diffusion area
of 1cm2 for 6 hours (20). The receptor phase was solution Tagat® CH 60 in water 0.5%
established based on a preliminary solubility study of terbinafine. The system was maintained
at 37±0.5°C for approximately 30 minutes before starting the experiment and during the
course of the same. The receptor medium was homogenized by small magnetic bar stirring.
The amount applied was 200-300 mg for either formulation (equivalent to 2000-3000 µg of
terbinafine). Samples of 200 µL were collected at predefined times (0, 1, 2, 3, 4, 5 and 6
hours) and the same volume was replaced with fresh receptor solution maintained at the same
temperature.
The samples of terbinafine were analysed by HPLC-UV in a Hitachi Elite Lachrom System
(VWR, USA) equipped with four Pumps L-2130, an autosampler L-2200, a column
Lichrospher 100 RP18, (150mm x 4mm, 5µm, Merck), an UV Detector L-2400 and signal
processing by software EZ Chrom Elite Version 3.2.1. The method used an isocratic gradient
mobile phase containing, 85% (v/v) methanol and 15% (v/v) solution of water 0.5%
triethanolamine. A flow rate of 1.0 mL/min was used with a 10 µL injection volume. The auto
sampler chamber was maintained at room temperature and the eluted peaks were monitored at
wavelength of 283 nm. The run time was 5 min. All chromatographic separations were carried
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out at 25ºC. A calibration curve was performed using 19.8 µg/mL; 39.7µg/mL; 79.4 µg/mL;
99.2 µg/mL and 119 µg/mL of terbinafine hydrochloride in methanol. Six replicate containers
for each formulation were analyzed.
2.2.9. In vitro antifungal activity
Candida albicans ATCC 10240 and Aspergillus brasiliensis ATCC 16404 were used for the
determination of in vitro antifungal inhibitory activity of a terbinafine and ciclopirox nail
lacquer formulations. The test was performed according to the standard disc diffusion method
(DDM) in accordance with the Clinical and Laboratory Standards Institute (CLSI)
guidelines25. The potato dextrose agar (PDA) (BioKar, Spain) and sabouraud dextrose agar
(SDA) (BioKar, Spain) microbiologic media were used for A. brasiliensis and for C. albicans,
respectively.
The test took place over 2 days under 37 ± 2 ºC for C. albicans and 7 days under 22.5 ± 2.5
ºC for A. brasiliensis. The positive or negative activity of the formulation was assessed
according to the presence or absence of growth around the disks and is an indirect measure of
the ability of the formulation to inhibit those microorganisms. A. brasiliensis inoculum was
prepared from 5–7 days old cultures grown on Mueller-Hinton agar (BioKar, Spain). The
suspensions obtained were diluted with distilled water to obtain a turbidity equivalent to a
McFarlard 0.5 turbidity standard25, containing 3.4 x 108 cfu/mL of microorganisms. C.
albicans inoculum was prepared from 1 day cultures grown on Mueller-Hinton agar. The
suspensions obtained were diluted with distilled water to obtain a turbidity equivalent to a
McFarlard 0.5 turbidity standard25, containing 7.2 x 108 cfu/mL of microorganisms. Sterile
paper discs (6 mm, Sigma-Aldrich, UK), were loaded with 15 µL of each formulation.
Placebo formulations were used to compare the inhibition zones. All the results were recorded
by measuring the zone of growth inhibition surrounding the discs. All determinations were
made in duplicate.
2.2.10. Final formulations - Permeation studies
The nail tips were obtained by cutting the free edge of the nail plate of healthy volunteer
(female 25 years old) after ethical approval and informed consent. The samples had a
minimum length of 5 mm. The nail tips were hydrated in 10 mL receptor solution (some in
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aqueous solution of 0.5% Tagat® CH 60 and the other in phosphate buffer saline to which
sodium azide) for 1 h. Ony-Tec® was used as control. The nail tip samples were sandwiched
between two cylindrical adapters made of polytetrafluoroethylene (PTFE) (Mecanizados del
noroeste, Santiago de compostela, Spain) with an o-shaped ring providing an effective
diffusional area of 0.049 cm2. The set was placed between the donor and receptor chambers of
vertical Franz-type diffusion cell (Vidrafoc, Barcelona, Spain) of the dorsal and ventral layers
of the nails faced the donor and the receptor compartments, respectively. The donor chamber
contained either 2 ml of one of the nail lacquer (Formulation A PU19-10% TH, Formulation
G PU19 10% CPX) and was covered with parafilm®. The receptor medium, 5.5 mL receptor
volume, was aqueous solution of 0.5% Tagat® CH 60 for Formulation A PU19-10% TH and
phosphate buffer saline which sodium azide (30 mg/L) pH 7.4 (9) for Formulation G PU19-
10% CPX and Ony-Tec®. The receptor compartments were at constant temperature by use of
thermostated (32± 0.5 oC) water. The sink conditions were assumed. Samples of 1000 µL
were collected at predefined times (each day at same time) and the same volume was replaced
with fresh receptor solution maintained at the same temperature. The experiments were
performed for 11 days and three replicates were made for each condition (9).
The amount of TH and CPX present in the nail plate at the end of penetration experiment was
also determined by UV spectrophotometry (spectrophotometer diode array Hewlett Packard
8452A). The wavenumber for terbinafine was 283 nm (21) and for ciclopirox 308 nm (9) after
the drug extraction. The section of the nail exposed to the formulation was cut, weighed and
cut in small fragments that were transferred into a vial containing either 5 ml of receptor
solution at 5% in methanol. Later, the prepared solutions were incubated and shaken for 4
days at room temperature to facilitate drug extraction. The CPX extraction method was
adapted and validated from the literature (9,21) while the TH extraction method was validated
for sensitivity, linearity and precision in the concentration range of 6.32 µg/mL-31.6 µg/mL
(r2=0.9884, calibration curve standards were assayed together with every sample batch to
account for inter day variability).
Standard and blank solutions were made up using PBS buffer (pH 7.4) containing 30 mg/L of
sodium azide and aqueous solution of 0.5% Tagat® CH 60.
The data were normalized to account for thickness of the nails.
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2.2.11 Porosity measurement
Untreated and treated nail samples were analyzed using a Micromeritics 9305 pore sizer
(Norcross GA, USA) fitted with a 3-mL powder penetrometer and working pressures in a
0.004–172.4 MPa range. Nail tips were used as described in chapter 5, section 2.2.10.
The nails were soaked in 10 mL of either water Ony-Tec and Formulation A PU19-10% TH.
Later the samples were stored at room temperature for 24h. After the time, the nails were
removed from the solution tested, dried and determined the porosity. The amount of nail tip
for test was approximately 0.6 g. The pore size data were used for modeling the porous
structure of the samples as simulated porous networks using poreXpert™ 1.3 software
(Environmental and Fluid Modelling Group, University of Plymouth, UK)(22).
3. Results and discussion
3.1 Direct contact assay
The cell viability test was carried out for nail lacquer containing different percentages of
polymers and control slide glass. Phase contrast micrographs were taken at the interface of the
cell layer with outer contact areas of the different matrices.
In the Figure 5.1, were observed the color of HaCaT cells in contact with MTT. The purple
color was detected in the almost every samples, confirming the viability of cell in contact with
all the formulations. These results corresponding to the measurement of PU19 section 3.9,
chapter 3.
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Figure 5.1. Micrograph with 400x magnification of HaCaT cells after 72h of proliferation
under contact with different therapeutic nail lacquer: A) glass slide, B) Formulation A PU19-
10% TH, C) Formulation D PU19-15% TH, D) Formulation E PU19-20% TH, E)
Formulation F PU19-25% TH and F) PU19.
Figure 5.2 represents the percent of cell viability measured by the MTT reduction for the nail
lacquer formulations.
Figure 5. 2. HaCaT cell viability by MTT after proliferation under different nail lacquers.
Control (glass slide), (mean ± SD, n = 6).
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From Figure 5.2 it can be observed that PU 19, Formulation A PU19-10% TH, Formulation D
PU19-15% TH, Formulation E PU19-20% TH and Formulation F PU19-25% TH presents a
cell viability (measured by the MTT reduction) that did not differ significantly from the
control (glass slide) and PU19 (section 3.9, chapter 3). The polymer concentration did not
influence the cell viability.
The Formulation G PU19-10% CPX cell viability is 12% (significantly different from control
for p<0.05). This formulation is not biocompatible and not allow cell growth.
The results of cell viability of PU19-10% CPX probably present relation to the proportion of
ethyl acetate and the ciclopirox. Ethyl acetate is used as a solvent for chemical reactions and it
is often used in cosmetics as nail polishes because is not toxic but in short-term exposure to
high levels of ethyl acetate results in irritation of different tissues (23)
All the samples tested with TH allow the cell proliferation under the surface covered by the
polymers that was confirmed by the presence of a monolayer of viable cells.
The Formulation G PU19-10% CPX, will be study in the rest of the chapter only with
comparative objective.
3.2 Wettability
As referred previously (section 3.2, chapter 4), the wettability tests show that the water
contact angle of PU19 based nail lacquer is lower than the one observed for the nail surface
alone, which presented an average value of 94º, indicating that the nail lacquer favors the
wetting of the surface. However, different concentrations of the polymer in the solutions that
were used to prepare the films led to variations in the water contact angle. An increase in the
content of PU19 origins higher contact angles: while for 10% of PU19 a water contact angle
of 45º4º is obtained, for 20% of PU19 it increased to 66º4º and for 25% to 68º11º (Figure
5.3). This fact shall be related with the surface morphology of the films, since the chemical
composition is the same after the evaporation of the solvent. As it can be observed in SEM
images (see below, Figure 5.4), the surfaces of Formulation A PU19-10% TH are more
irregular than those produced from solutions with higher contents of polymer. This behavior
may be explained by the Wenzel theory(24). According to this author, the equilibrium contact
angle in a rough surface (W, apparent Wenzel angle) is related with the real contact angle ()
trough Equation 1:
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cos W = r cos (1)
where r is a roughness factor, defined as the reason between the real surface area and the
corresponding projected area. This approach implies that an increase of roughness leads to a
decrease of the observed contact angle when W is lower than 90º, as is the case.
Figure 5.3.Water contact angle of PU terbinafine nail lacquers (mean ± SD, n= 3).
Wettability measurements were also carried out with samples PU19-10% loaded with CPX,
instead of TH. The water contact angle was similar in both cases (45º4º and 42º4º,
respectively), being visible that the surface morphologies are similar (Figure 5.4 below). This
means that the nature of the drug does not affect the wettability of the surfaces.
Overall, these results allow to select Formulations A PU19-10% and G PU 19-10% CPX as
those which present better wettability properties.
3.3 SEM
The images obtained by SEM of nail lacquers show homogeneous films, which cover the
surface to the nail as observed in Figure 5.4.
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Figure 5.4. Scan Electron Micrograph with 1000x magnification. A) dorsal surface of nail
plate B) film of Formulation A PU 19-10% TH, C) film of Formulation D PU19-15% TH, D)
film of Formulation E PU19-20% TH, E) film of Formulation F PU19-25% TH, F) film of
Formulation G PU19-10% CPX and G) Ony-Tec®.
The inclusion of the drug (TH or CPX) had no influence on film thickness and on the film
thickness and on the microstructure of PU based nail lacquers. However, Ony-Tec® did not
form a homogenous film in the surface of the nail. These measurements indicated that the
application of the PU nail lacquers coated the roughness of the nail surface, making it smooth
and uniform, while chitosan film from (9) was similar to the untreated nail with visible pores
and cracks on the surface.
3.4 Adhesion test results
The nail lacquer must to adhere to nail. The duration of residence of the film on the nail plate
is therefore critically important, because the film of nail lacquer acts as a drug depot, from
which the drug can be continuously released and permeate into the nail. A film with a long
residence time would need less frequent lacquer application, which could in turn lead to
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increased patient compliance, improved treatment efficacy and reduced cost of treatment (25).
In the Figure 5.5 are presented the evaluation of adhesion test.
Figure 5.5. Nail lacquer adhesion test to keratin of cow horn.
All the formulations presented adhesion to keratin of cow horn, as observed in Figure 5.5. The
adhesion values in cow horn corresponding to better results in compared with the result of
adhesion from nail lacquer formulated with methacrylates, where the cross cut mean score
were between 4 and 5 (10).
On the other hand, the nail lacquer that presents the higher flaking in the lattice pattern was
formulation Ony-Tec®. The formulation Ony-Tec® is a chitosan derivative’ hydrogel.
Previously study by SEM showed a film with pores and cracks on the surface of nail,
furthermore hidroypropyl chitosan is a water soluble film former with affinity to keratin(26).
The results obtained permit to concluded that the polyurethanes films present more affinity to
keratin that Ony-Tec®.
The formulation with more adhesion to cow horn was the Formulation G PU 19-10% CPX,
with a degree of flaking in the lattice pattern of 0.1, probably, because the formulation G
PU19-10% presented highest value ethyl acetate as solvent and permit more exposition of the
hydrophilic group of polyurethane to keratin of cow horn (27).
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3.5 Nail lacquer’s drying time
For complaint of patient the formulation must dry in the nail (29–32). In table 5.2 is observed
the value of drying time of formulation.
Table 5. 2. Nail lacquer’s drying time (mean±SD, n= 3).
Formulation Time (min)
Formulation A PU19-10% TH 9±0.1
Formulation D PU19-15% TH 10±0.3
Formulation E PU 19-20% TH 15±0.1
Formulation F PU19-25% TH 16±0.2
Formulation G PU19-10% CPX 7±0.1
Ony-Tec® 13±0.1
The PU formulations present drying times from 9 to 16 min. In fact, the higher the
concentration of the polymer, the longer the drying time of the nail lacquer. These results are
in agreement with the solvent contents because the higher the concentration of ethyl and butyl
acetate the shorter the drying time.
The Ony-Tec® presents a drying time of 13 minutes, which is related to its composition.
Ethanol is main solvent of this formulation. According to these results, Formulation A PU19-
10% TH, Formulation D PU19-15% TH and the Formulation G PU19-10% CPX should be
selected.
3.6 Viscosity values
The values of viscosity for the PU terbinafine nail lacquers at 32oC in order to mimic nail
lacquer application are shown in Table 5.3. It observed that the higher the concentration of
polymer increased the viscosity.
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Table 5. 3. Viscosity values of therapeutic nail lacquers (mean±SD, n= 3).
Formulation Viscosity (mPa.s)
Formulation A PU19-10% TH 2.62±0.04
Formulation D PU19-15% TH 4.70±0.06
Formulation E PU19-20% TH 8.80±0.11
Formulation F PU19-25% TH 17.03±0.07
Formulation G PU19-10% CPX 2.24±0.04
Ony-Tec® 5.47±0.06
As already explained in chapter 4, formulations that presents more reticulated network (a gel)
do not allow a high release of the drug. Thus, and using Ony-Tec® as a reference with regard
to viscosity values, Formulation A PU19-10% TH, Formulation D PU19-15% TH and
Formulation G PU19-10% CPX are the ones that, probably should release the amount of drug
needed to be effective (35).
3.7 Preliminary test for select formulation with better condition to release the
terbinafine
In order to confirm that the polymer concentration affect the drug release an in vitro
preliminary test was assessed just using the TH formulations. The results confirm that the
highest the polymer concentration the less amount of drug is released (Figure 5.6).
0 1 2 3 4 5 60
10
20
30
40
50
60
70
80
90
100
PU19-25%
PU19-20%
PU19-15%
PU19-10%
Time (hours)
Te
rbin
afin
e re
lea
se
(%
)
Figure 5.6. Release of TH from different formulations at 32 ºC (mean ± SD, n = 6).
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The Formulation A PU19-10%TH release 66.96%, after 6h, while the rest of formulation
retain the terbinafine in the matrix for the same time of study. The Formulation D PU19-
15%TH release 16%, the Formulation E PU19-20%TH release 10.6% and the Formulation F
PU19-25%TH release 6%. These results indicated that the increase of amount of polymer,
decreases the release of terbinafine from the formulations.
In another study with the aim of investigate the role of hydrophilic polymers in sustaining the
release of drugs in tablets dosages form, was find that the higher viscosity, have relation with
resistance of matrix to dissolution and erosion. It was study that matrix prepared with hydroxy
propyl methyl cellulose, present more viscosity of hydroxyethyl cellulose, this increase of
viscosity could explain the release retardation property of the hydroxy ethyl cellulose (36).
Furthermore, the movement of the drug solute through the matrix system is diffusion
controlled, it may be expected that the dissolution process will be slower in the more viscous
medium (35).
3.8 Antifungal activity
The antifungal activity of the drugs against Candida albicans ATCC 10240 and Aspergillus
brasiliensis ATCC 16404 were performed. The results are shown on Table 5.4.
The terbinafine present antifungal activity against Candida albicans(37) however the CPX is
more effective against this yeast(18).
On the other hand the CPX present lower activity against Aspergillus brasiliensis(18).
Table 5. 4. Inhibition zone (mm) of all formulations in plate dish for Candida albicans, and
Aspergillus brasiliensis (mean±SD, n=2).
Inhibition zone (mm)
Formulation A
PU19 10% TH
Formulation D
PU19 15% TH
Formulation E
PU19 20% TH
Formulation F
PU19 25% TH
Formulation G
PU19 10% CPX
Solution
TH(1%)
Solution
CPX(1%) Ony-Tec®
Candida
albicans
ATCC 10240
38.4±3.6 29.7±0.4 29.0±0.6 26.2±1.0 32.3±1.3 21.4±1.8 29.0±1.1 43.9±2.0
Aspergillus
brasiliensis
ATCC 16404
25.0±0.4 23.7±0.6 23.2±0.1 21.1±1.7 26.7±0.3 32.0±0.8 31.4±0.4 33.3±0.4
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All the nail lacquer formulation presents antifungal activity against the fungi of the study.
The results obtained demonstrated the nail lacquer formulations with higher antifungal
activity, are the ones that have the lower polymer concentration probably, because they allow
a higher release of the drug. All the control formulation presented inhibition zone < 6mm.
The solution TH formulation presented higher inhibition zone against Candida albicans
probably for the influence of -NH- of polyurethane in the pH of the medium that potential the
antifungal activity of the drug. The in vitro activity of terbinafine is pH dependent and rises
with increasing pH value (38).
The Formulation G PU19-10% CPX present one interesting value of antifungal activity in
comparison with Ony-Tec®.
3.9 Permeation Test
Know the amount of drug that permeates the nail in an animal model similar to that of man,
for purposes of pre-clinical studies is still under study and to date has not been established.
The optional solution to be able to study the passage of the drug is the use of healthy human
nails (4,39,40).
Permeation profiles of drugs into the nail are shown in Table 5.5. As it was observed, in
release experiments (section 3.6), the presence of polyurethane allows the diffusion of
terbinafine trough hydrophilic membrane and as expected the results of this study was similar.
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Table 5. 5. Permeation profiles of formulations and Ony-Tec® trough the nail. (mean±SD,
n=3).
Time
(h)
Amount of Drug Diffusion (µg/cm3)
Formulation A
PU 19 10% TH
Formulation G
PU19 10% CPX Ony-Tec
24 1542.24 ± 896.73 561.31 ±482.34 282.53 ±53.70
48 4030.43 ± 3934.95 3502.19 ± 2639.37 290.53 ±43.50
72 3809.94 ± 3435.92 3695.06 ± 2829.67 342.53 ±43.80
96 3899.71 ± 3386.31 3850.59 ± 2938.92 582.53 ±43.70
120 4273.13 ± 3659.95 3992.68 ± 2929.31 540.53 ±33.60
168 4405.92 ± 3714.03 3691.65 ±2627.53 560.53 ±23.70
192 4236.98 ± 3488.06 3723.40 ±2640.22 550.53 ±43.80
216 4214.38 ± 3343.91 3810.57± 2671.87 586.53 ±53.20
240 4389.38 ± 3454.15 3910.11±2785.01 593.53 ±67.20
264 4435.47 ± 3391.68 3909.61 ± 2769.71 598.53 ±33.20
The values of drugs permeated trought the nail from Formulation A PU19-10% TH after 11
days was 4435.47 µg/cm3 and from Formulation G PU19-10% CPX was 2649 µg/cm3. The
results showed that the total amount of TH permeated trought the nail was higher than the
CPX and Ony-Tec®.
The permeated profiles of both drugs indicate that the higher concentrations in receptor
solution are obtained in the first 120 hours after application, probably attributed to the
presence of ethanol in the tested formulation. The ethanol increased the drug solubility in the
vehicle and has action as enhancer (41) . Furthermore, the small molecular size of both drugs
allows the permeation through the nail (TH-291.43 g/mol and CPX-207.3g/mol)(5).
Regarding to standard deviation find in all the formulation testing is possible to conclude that
in the permeation process influence: the thickness of nail that if different from one individual
to another and the composition of nail (percent of water, lipid, mineral)(42) and drug-keratin
interactions(43).
In another research where were testing the diffusion coefficient of amorolfine, terbinafine and
caffeine, the standard deviation was significates. The author explains that the data which were
not corrected in terms of nail thickness, however the number of replicated were n=5-7 for
caffeine, n=11-13 for the amorolfine and n=13-15 for terbinafine did not prevent the finding
of equal and superior deviations from the determination(4).
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Then the quantities of each drug were determinate in the nail plate. Results are shown in
Figure 5.7.
Figure 5.7. Amount of drug determined in nail plate after 11 days of experiments. One-way
ANOVA and Tukey’s multiple comparison tests indicates significant differences between
Ony-Tec® and PU based nail lacquer formulations.
The amount of CPX and TH determined in nail plate, showed similar values, furthermore the
formulations based polyurethane retain more drug in keratin structure that Ony-Tec®,
probably to the concentration of ethanol as vehicle in the formulation.
The amount of TH determined in nail presented higher values (>0.004 -1 µg/mL), value of
minimum inhibitory concentration of TH against Candida albicans and Aspergillus
species(18).
On the other hand the values of CPX determined in nail are superior to the range 0.015- 4
µg/mL corresponding to minimum inhibitory concentration of drug, against Candida albicans
and Aspergillus species respectively (18).
3.10 Porosity study
The porosity of the nail is another parameter to take in count for explain changes in the
diffusion of drug due to modification on hydration of nail plate. This hydration allows drug
flux from certain vehicles (22). Indeed, an increase in nail plate hydration was found to
increase the permeation ketoconazole by three-fold and the diffusivity of water by more than
400-fold, when relative humidity was increased from 15 to 100% (36). One method useful to
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characterize the internal microstructure of the nail is mercury intrusion porosimetry (MIP) in
combination with PoreXpert™ software. The combination of these techniques allow to obtain
models useful to characterize the permeability properties of the nails (22). In the figure 5.8 are
presented the values of porosity of nail obtained by MIP before and after different treatment.
a) b) c)
Figure 5.8. Cumulative curves of porosity obtained by MIP for treated and untreated nail and
PoreXpert models of the microstructrure of a) untreated nail, b) Ony-Tec® treated nails and c)
Formulation A PU19-10% TH treated nails. Cubes represent the pores and cilinders the
conection between pores.
No significant changes on the microstructure of the nail were observed after the application of
nail lacquers. The cumulative pore distribution curve of Ony-Tec® seems to be relatively
higher for lower pore diameter but the model and the parameters characteristic of the model
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obtained by PoreXpertTM (table 5.6) indicates similar porosity and connectivity regardless the
formulation. The values of correlation of the models, near the unity, obtained in treated and
untreated nail indicates structures with high level order caused by the well delimited areas
with high porosity at the top and bottom of the model and the low porosity structure in the
middle of the model. Similar results were obtained by Nogueiras et al (9) for healthy
untreated nail using MIP plus PoreCoreTM software.
These results indicates that Formulation A PU19-10% TH and Ony-Tec® did not produce
microstructural changes in the nail plate probably due to the presence of organic solvents and
PU19-10% or the low proportion of water (Ony-Tec®) used in the elaboration of these nail
lacquers.
Table 5. 6. Main parameters of the models obtained from porosity data using PoreXpert.
Porosity
(%) Correlation Conectivity Water permeability
(mD)
Untreated nails 6.83 % 0.795 5.02 1.5775x10-6
Ony-Tec® 6.75 % 0.796 5.02 1.5417x10-6
Formulation A
PU19-10%TH 6.75 % 0.795 5.02 1.5775 x10-6
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4. Conclusions
This study reports the physic-chemical characterization of TH and CPX nail lacquer
formulations based polyurethane. The TH nail lacquers obtained were compatible with
keratinocytes cell and presented hydrophilic properties, however the CPX nail lacquer
presented incompatible with skin cell.
While the homogenous films obtained from the nail lacquer presented adhesion to nail. The
viscosity studied demonstrated that the Formulation A PU19-10% TH and Formulation G
PU19-10% CPX presented low viscosity compared to Ony-Tec®.
In vitro release studies reveal that the amount of PU19 used had influence on the release
profiles of drug, which was in agree with the antifungal activity of the formulations.
All the nail lacquer formulations showed antifungal activity against Candida albicans and
Aspergillus brasiliensis. The permeation profiled of antifungal formulation through the nail
were higher that Ony-Tec®.
The result obtained of permeation and determination of CPX in nail allow to prepare another
formulation with used of PU19 with the aims to improve the compatibility with keratinocytes
cells.
The use of polyurethane in nail formulation can be a promising strategy for the release of
lipophilic drug.
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Chapter 6: Conclusions and Final Remarks
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Conclusions and Near-Future Research
Synthesis of polyurethanes employing isosorbide and aliphatic diisocyanate. Four
polyurethanes (PU) obtained from isosorbide, PPG and aliphatic diisocyanate were
synthesized and characterized. The soft segment segmentation (PPG) increased the molecular
weight and the hydrodynamic radius of PU but decreased the diffusion coefficient of the
macromolecules. On the other hand, increasing the extender of chain (isosorbide) the PU
adhesion to keratin gave better results. The polyurethane presented adequate solubility
properties.
According to these properties and diffusion coefficient, determined by NMR, polyurethane 19
(PU19), synthesized from 1 mol PPG, 6 mol IPDI and 5 mol of isosorbide, was selected for
the final formulations.
Further investigation may include:
Experimental design using other molar relation and different temperatures should be assessed
with this monomer.
Determination of polymers’ molecular weight in different solvents according to
hydrodynamic volume should also be performed.
PU based nail lacquers: formulation and antifungal activity evaluation.
PU-terbinafine based nail lacquers prepared with four PU selected showed keratinocyte
compatibility, good wettability properties and adequate free volume. They formed a
homogenous film after application, with suitable adhesion to the nail plate. Furthermore, the
antifungal test results demonstrated that the terbinafine released from the nail lacquer
Formulation A PU 19 showed activity against dermatophytes namely, Tricophyton rubrum.
Further investigation may include:
Structural studies using Wide- Angle X-ray Scattering (WAXS), Atomic Force Microscopy
(AFM) and Differential Scanning Calorimetry (DSC) techniques would complement the
physicochemical characterization of the PU based nail lacquers.
Transungueal permeation studies
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Preparation of 19 PU based nail lacquer.
PU 19 was the polymer selected to prepared four nail lacquers with different amounts of
polymer, 10%, 15%, 20% and 25%. The concentration of 10% permitted a nail lacquer with
better adhesion properties, release and antifungal results.
Further investigation may include:
Study of the effect of lower quantities of polyurethane in order to determine the best nail
lacquer formulation with drug effectiveness.
Formulation optimization of PU based Nail Lacquers containing Terbinafine
hydrochloride and Ciclopirox Olamine.
The incorporation of two drugs (TH and CPX) validated the use of PU as versatile polymers
in nail lacquer formulations. Direct contact cytotoxicity, wettability, SEM, adhesion, drying
time, viscosity measurement, permeation studies and antifungal activity were performed.
The results indicates that the total quantity of terbinafine hydrochloride permeated trought the
nail was higher than the ciclopirox from the same matrix. The developed nail lacquers
permitted better diffusion of antifungal drugs through the nail that reference one (Ony-Tec®).
The amount of terbinafine hydrochloride and ciclopirox olamine determined in nail were
higher than the minimum inhibitory concentration of these drugs against Candida Albicans
and Aspergillus brasiliensis. However, CPX formulation was incompatible to keratinocytes
cells. The antifungal activity of the terbinafine hydrochloride and ciclopirox olamine PU
based nail lacquer against Candida albicans and Aspergillus brasiliensis was determined. The
dresults obtained demonstrated that the amount of drug release from the nail lacquer
formulations presented antifungal activity. The influence of -NH- of polyurethane in the pH
of the medium, potential the antifungal activity of the terbinafine hydrochloride against
Candida albicans
Further investigation may include:
Diffusion and permeation studies in keratin should be performed with TOWL determination
in each nail. The development of new safe and biocompatible formulations with CPX
effectiveness must be performed.
Stability test should be done.
Antifungal activity against dermatophytes and non dermatophytes.
Clinic trials should be assessed.
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The research work presented in this thesis, led to the development of a new nail lacquer with
adhesive, biocompatible and improved the antifungal activity.
Nevertheless, this new nail lacquers (polyurethane-antifungal drugs) and taking into account
the challenge of treating nail infections, need further studies such as structural and clinical
studies.
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Annexes
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201
Annex 1. Spectrum 1H NMR PEG 1 500
ppm 0.05.0
7.2
6
3.8
6
3.8
5
3.8
3
3.6
4
3.6
3
3.6
1
3.3
9
3.3
7
2.7
4
2.5
0
1.2
2
0.0
3
New polyurethane nail lacquers for the delivery of antifungal drugs Annexes
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Annex 2. Spectrum 1H NMR pMDI
ppm 1.02.03.04.05.06.07.08.0
7,1
8
7,1
3
7,1
0
7,0
8
7,0
6
7,0
3
7,0
1
6,9
9
6,9
6
6,9
3
6,9
0
6,8
8
6,7
9
6,7
7
3,9
2
3,8
8
3,8
6
3,2
9
3,2
9
2,9
6
1,5
0
1,5
0
1,2
1
0,0
3
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Annex 3. Spectrum 1H NMR Sucrose
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204
Annex 4. MALDI-TOF mass spectra of PU19
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205
Annex 5. MALDI-TOF mass spectra of PU20
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Annex 6. MALDI-TOF mass spectra of PU21
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Annex 7. MALDI-TOF mass spectra of PU22